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Dysferlin-deficient immortalized human myoblasts and myotubes )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(as a useful tool to study dysferlinopathy. PLOS Currents Muscular Dystrophy. 2012 Feb 28 . Edition 1. doi: )] TJ ET BT 26.250 627.533 Td /F1 9.8 Tf [(10.1371/currents.RRN1298.)] TJ ET q 15.000 22.378 577.500 602.774 re W n 0.271 0.267 0.267 rg BT 26.250 598.430 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(Dysferlin gene mutations causing LGMD2B are associated with defects in muscle membrane repair. Four stable cell lines have )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(been established from primary human dysferlin-deficient myoblasts harbouring different mutations in the dysferlin gene. We )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(have compared immortalized human myoblasts and myotubes carrying disease-causing mutations in dysferlin to their wild-type )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(counterparts. Fusion of myoblasts into myotubes and expression of muscle-specific differentiation markers were investigated )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(with special emphasis on dysferlin protein expression, subcellular localization and function in membrane repair. We found that )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(the immortalized myoblasts and myotubes were virtually indistinguishable from their parental cell line for all of the criteria we )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(investigated. They therefore will provide a very useful tool to further investigate dysferlin function and pathophysiology as well )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(as to test therapeutic strategies at the cellular level.)] TJ ET BT 26.250 458.540 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 438.586 Td /F1 9.8 Tf [(The Deutsche Forschungsgemeinschaft funded the study through Myograd \(GK1631\) and the clinical research group KFO192 )] TJ ET BT 26.250 426.681 Td /F1 9.8 Tf [(Skeletal muscle growth regulation and dysregulation. The Jain Foundation and the Association Franaise contre les )] TJ ET BT 26.250 414.777 Td /F1 9.8 Tf [(Myopathies \(AFM\) also supported this investigation.)] TJ ET BT 26.250 385.674 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(Muscular dystrophies comprise clinically and genetically heterogeneous disorders characterized by progressive weakness and )] TJ ET BT 26.250 353.815 Td /F1 9.8 Tf [(wasting of the skeletal muscle accompanied by an increase in muscle connective tissue)] TJ ET 0.267 0.267 0.267 rg BT 403.945 353.815 Td /F1 9.8 Tf [( [1])] TJ ET 0.271 0.267 0.267 rg BT 417.498 353.815 Td /F1 9.8 Tf [( Dysferlin gene mutations cause limb )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(girdle muscular dystrophy 2B \(LGMD2B\) and Miyoshi myopathy, allelic autosomal recessive diseases characterized by limb )] TJ ET BT 26.250 330.006 Td /F1 9.8 Tf [(girdle or distal weakness of early adult onset )] TJ ET 0.267 0.267 0.267 rg BT 220.792 330.006 Td /F1 9.8 Tf [([2] [3])] TJ ET 0.271 0.267 0.267 rg BT 245.186 330.006 Td /F1 9.8 Tf [( . Dysferlin \(MIM*603009\) is a 230kDa transmembrane protein comprising )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(calcium binding C2 domains that is highly expressed in skeletal muscle )] TJ ET 0.267 0.267 0.267 rg BT 335.111 318.101 Td /F1 9.8 Tf [([4] [5] )] TJ ET 0.271 0.267 0.267 rg BT 362.216 318.101 Td /F1 9.8 Tf [(. Dysferlin localizes to the sarcolemma and is )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(involved in membrane repair, membrane trafficking and muscle regeneration )] TJ ET 0.267 0.267 0.267 rg BT 358.413 306.196 Td /F1 9.8 Tf [([6] [7] [8])] TJ ET 0.271 0.267 0.267 rg BT 396.360 306.196 Td /F1 9.8 Tf [( . Various mutations associated with )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(LGMD2B have been identified in dysferlin. These mutations lead either to a reduced expression of dysferlin at the sarcolemma, )] TJ ET BT 26.250 282.387 Td /F1 9.8 Tf [(an intracelluar accumulation of dysferlin, the formation of amyloid-like deposits or to the complete absence of dysferlin protein )] TJ ET BT 26.250 270.482 Td /F1 9.8 Tf [(finally resulting in impaired muscle membrane repair )] TJ ET 0.267 0.267 0.267 rg BT 254.351 270.482 Td /F1 9.8 Tf [([9] [10] [11])] TJ ET 0.271 0.267 0.267 rg BT 303.140 270.482 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 251.077 Td /F1 9.8 Tf [(The access to primary human myoblasts from biopsies of patients with disease-causing dysferlin mutations is limited. Due to )] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(excessive fibrosis, these muscle biopsies often contain only very few myogenic cells and are highly intermingled with connective )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(tissue cells like fibroblasts and adipocytes. Additionally, primary human myoblasts in culture show a limited proliferative potential )] TJ ET BT 26.250 215.363 Td /F1 9.8 Tf [(and undergo changes that are linked to replicative senescence)] TJ ET 0.267 0.267 0.267 rg BT 296.686 215.363 Td /F1 9.8 Tf [( [12])] TJ ET 0.271 0.267 0.267 rg BT 315.659 215.363 Td /F1 9.8 Tf [( . To circumvent these limitations immortalized human )] TJ ET BT 26.250 203.458 Td /F1 9.8 Tf [(myoblast lines were generated by retroviral transduction of primary human myoblasts harbouring different disease-causing )] TJ ET BT 26.250 191.553 Td /F1 9.8 Tf [(mutations with telomerase \(hTERT\) and cyclin-dependent kinase 4 \(CDK-4\). The expression of hTERT overcomes the )] TJ ET BT 26.250 179.649 Td /F1 9.8 Tf [(progressive erosion of telomeres occuring due to cell division and the overexpression of CDK-4 blocks the induction of the p16-)] TJ ET BT 26.250 167.744 Td /F1 9.8 Tf [(mediated cellular stress-pathway )] TJ ET 0.267 0.267 0.267 rg BT 170.921 167.744 Td /F1 9.8 Tf [([13])] TJ ET 0.271 0.267 0.267 rg BT 187.184 167.744 Td /F1 9.8 Tf [( . After their immortalization these cell lines show a prolonged proliferation and )] TJ ET BT 26.250 155.839 Td /F1 9.8 Tf [(differentiation capacity compared to primary human myoblasts )] TJ ET BT 297.183 155.839 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 325.897 155.839 Td /F1 9.8 Tf [( and they can be transplanted into regenerating muscle )] TJ ET BT 26.250 143.934 Td /F5 9.8 Tf [(in vivo )] TJ ET 0.267 0.267 0.267 rg BT 56.592 143.934 Td /F1 9.8 Tf [([13])] TJ ET 0.271 0.267 0.267 rg BT 72.855 143.934 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 124.530 Td /F1 9.8 Tf [(Here we describe a detailed analysis of immortalized human myoblast lines harbouring different dysferlin mutations. These cell )] TJ ET BT 26.250 112.625 Td /F1 9.8 Tf [(lines maintain the characteristics of primary dysferlin-deficient human cell strains with respect to myogenic differentiation, )] TJ ET BT 26.250 100.720 Td /F1 9.8 Tf [(dysferlin expression and membrane repair and represent a useful tool to further investigate all aspects of dysferlin function and )] TJ ET BT 26.250 88.815 Td /F1 9.8 Tf [(dysfunction.)] TJ ET BT 26.250 52.213 Td /F4 12.0 Tf [(Material and Methods)] TJ ET Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Dysferlin-deficient immortalized human myoblasts and )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(myotubes as a useful tool to study dysferlinopathy)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(February 28, 2012)] TJ ET BT 93.263 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 98.138 675.088 Td /F3 9.8 Tf [(Methods)] TJ ET 0.271 0.267 0.267 rg BT 26.250 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 31.671 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 37.092 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 42.513 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 47.934 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 53.355 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 58.776 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 64.197 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 69.618 663.247 Td /F1 9.8 Tf [(Susanne Philippi)] TJ ET 0.271 0.267 0.267 rg BT 142.236 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 147.657 663.247 Td /F1 9.8 Tf [(Anne Bigot)] TJ ET 0.271 0.267 0.267 rg BT 195.354 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 200.775 663.247 Td /F1 9.8 Tf [(Andreas Marg)] TJ ET 0.271 0.267 0.267 rg BT 262.005 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 267.426 663.247 Td /F1 9.8 Tf [(Vincent Mouly)] TJ ET 0.271 0.267 0.267 rg BT 328.656 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 334.077 663.247 Td /F1 9.8 Tf [(Simone Spuler)] TJ ET 0.271 0.267 0.267 rg BT 398.017 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 403.438 663.247 Td /F1 9.8 Tf [(Ute Zacharias)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Philippi S, Bigot A, Marg A, Mouly V, Spuler S, Zacharias U. Dysferlin-deficient immortalized human myoblasts and myotubes )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(as a useful tool to study dysferlinopathy. PLOS Currents Muscular Dystrophy. 2012 Feb 28 . Edition 1. doi: )] TJ ET BT 26.250 627.533 Td /F1 9.8 Tf [(10.1371/currents.RRN1298.)] TJ ET q 15.000 22.378 577.500 602.774 re W n 0.271 0.267 0.267 rg BT 26.250 598.430 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(Dysferlin gene mutations causing LGMD2B are associated with defects in muscle membrane repair. Four stable cell lines have )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(been established from primary human dysferlin-deficient myoblasts harbouring different mutations in the dysferlin gene. We )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(have compared immortalized human myoblasts and myotubes carrying disease-causing mutations in dysferlin to their wild-type )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(counterparts. Fusion of myoblasts into myotubes and expression of muscle-specific differentiation markers were investigated )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(with special emphasis on dysferlin protein expression, subcellular localization and function in membrane repair. We found that )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(the immortalized myoblasts and myotubes were virtually indistinguishable from their parental cell line for all of the criteria we )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(investigated. They therefore will provide a very useful tool to further investigate dysferlin function and pathophysiology as well )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(as to test therapeutic strategies at the cellular level.)] TJ ET BT 26.250 458.540 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 438.586 Td /F1 9.8 Tf [(The Deutsche Forschungsgemeinschaft funded the study through Myograd \(GK1631\) and the clinical research group KFO192 )] TJ ET BT 26.250 426.681 Td /F1 9.8 Tf [(Skeletal muscle growth regulation and dysregulation. The Jain Foundation and the Association Franaise contre les )] TJ ET BT 26.250 414.777 Td /F1 9.8 Tf [(Myopathies \(AFM\) also supported this investigation.)] TJ ET BT 26.250 385.674 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(Muscular dystrophies comprise clinically and genetically heterogeneous disorders characterized by progressive weakness and )] TJ ET BT 26.250 353.815 Td /F1 9.8 Tf [(wasting of the skeletal muscle accompanied by an increase in muscle connective tissue)] TJ ET 0.267 0.267 0.267 rg BT 403.945 353.815 Td /F1 9.8 Tf [( [1])] TJ ET 0.271 0.267 0.267 rg BT 417.498 353.815 Td /F1 9.8 Tf [( Dysferlin gene mutations cause limb )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(girdle muscular dystrophy 2B \(LGMD2B\) and Miyoshi myopathy, allelic autosomal recessive diseases characterized by limb )] TJ ET BT 26.250 330.006 Td /F1 9.8 Tf [(girdle or distal weakness of early adult onset )] TJ ET 0.267 0.267 0.267 rg BT 220.792 330.006 Td /F1 9.8 Tf [([2] [3])] TJ ET 0.271 0.267 0.267 rg BT 245.186 330.006 Td /F1 9.8 Tf [( . Dysferlin \(MIM*603009\) is a 230kDa transmembrane protein comprising )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(calcium binding C2 domains that is highly expressed in skeletal muscle )] TJ ET 0.267 0.267 0.267 rg BT 335.111 318.101 Td /F1 9.8 Tf [([4] [5] )] TJ ET 0.271 0.267 0.267 rg BT 362.216 318.101 Td /F1 9.8 Tf [(. Dysferlin localizes to the sarcolemma and is )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(involved in membrane repair, membrane trafficking and muscle regeneration )] TJ ET 0.267 0.267 0.267 rg BT 358.413 306.196 Td /F1 9.8 Tf [([6] [7] [8])] TJ ET 0.271 0.267 0.267 rg BT 396.360 306.196 Td /F1 9.8 Tf [( . Various mutations associated with )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(LGMD2B have been identified in dysferlin. These mutations lead either to a reduced expression of dysferlin at the sarcolemma, )] TJ ET BT 26.250 282.387 Td /F1 9.8 Tf [(an intracelluar accumulation of dysferlin, the formation of amyloid-like deposits or to the complete absence of dysferlin protein )] TJ ET BT 26.250 270.482 Td /F1 9.8 Tf [(finally resulting in impaired muscle membrane repair )] TJ ET 0.267 0.267 0.267 rg BT 254.351 270.482 Td /F1 9.8 Tf [([9] [10] [11])] TJ ET 0.271 0.267 0.267 rg BT 303.140 270.482 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 251.077 Td /F1 9.8 Tf [(The access to primary human myoblasts from biopsies of patients with disease-causing dysferlin mutations is limited. Due to )] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(excessive fibrosis, these muscle biopsies often contain only very few myogenic cells and are highly intermingled with connective )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(tissue cells like fibroblasts and adipocytes. Additionally, primary human myoblasts in culture show a limited proliferative potential )] TJ ET BT 26.250 215.363 Td /F1 9.8 Tf [(and undergo changes that are linked to replicative senescence)] TJ ET 0.267 0.267 0.267 rg BT 296.686 215.363 Td /F1 9.8 Tf [( [12])] TJ ET 0.271 0.267 0.267 rg BT 315.659 215.363 Td /F1 9.8 Tf [( . To circumvent these limitations immortalized human )] TJ ET BT 26.250 203.458 Td /F1 9.8 Tf [(myoblast lines were generated by retroviral transduction of primary human myoblasts harbouring different disease-causing )] TJ ET BT 26.250 191.553 Td /F1 9.8 Tf [(mutations with telomerase \(hTERT\) and cyclin-dependent kinase 4 \(CDK-4\). The expression of hTERT overcomes the )] TJ ET BT 26.250 179.649 Td /F1 9.8 Tf [(progressive erosion of telomeres occuring due to cell division and the overexpression of CDK-4 blocks the induction of the p16-)] TJ ET BT 26.250 167.744 Td /F1 9.8 Tf [(mediated cellular stress-pathway )] TJ ET 0.267 0.267 0.267 rg BT 170.921 167.744 Td /F1 9.8 Tf [([13])] TJ ET 0.271 0.267 0.267 rg BT 187.184 167.744 Td /F1 9.8 Tf [( . After their immortalization these cell lines show a prolonged proliferation and )] TJ ET BT 26.250 155.839 Td /F1 9.8 Tf [(differentiation capacity compared to primary human myoblasts )] TJ ET BT 297.183 155.839 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 325.897 155.839 Td /F1 9.8 Tf [( and they can be transplanted into regenerating muscle )] TJ ET BT 26.250 143.934 Td /F5 9.8 Tf [(in vivo )] TJ ET 0.267 0.267 0.267 rg BT 56.592 143.934 Td /F1 9.8 Tf [([13])] TJ ET 0.271 0.267 0.267 rg BT 72.855 143.934 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 124.530 Td /F1 9.8 Tf [(Here we describe a detailed analysis of immortalized human myoblast lines harbouring different dysferlin mutations. These cell )] TJ ET BT 26.250 112.625 Td /F1 9.8 Tf [(lines maintain the characteristics of primary dysferlin-deficient human cell strains with respect to myogenic differentiation, )] TJ ET BT 26.250 100.720 Td /F1 9.8 Tf [(dysferlin expression and membrane repair and represent a useful tool to further investigate all aspects of dysferlin function and )] TJ ET BT 26.250 88.815 Td /F1 9.8 Tf [(dysfunction.)] TJ ET BT 26.250 52.213 Td /F4 12.0 Tf [(Material and Methods)] TJ ET Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Dysferlin-deficient immortalized human myoblasts and )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(myotubes as a useful tool to study dysferlinopathy)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(February 28, 2012)] TJ ET BT 93.263 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 98.138 675.088 Td /F3 9.8 Tf [(Methods)] TJ ET 0.271 0.267 0.267 rg BT 26.250 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 31.671 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 37.092 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 42.513 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 47.934 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 53.355 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 58.776 663.247 Td /F1 9.8 Tf [(, )] TJ ET BT 64.197 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 69.618 663.247 Td /F1 9.8 Tf [(Susanne Philippi)] TJ ET 0.271 0.267 0.267 rg BT 142.236 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 147.657 663.247 Td /F1 9.8 Tf [(Anne Bigot)] TJ ET 0.271 0.267 0.267 rg BT 195.354 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 200.775 663.247 Td /F1 9.8 Tf [(Andreas Marg)] TJ ET 0.271 0.267 0.267 rg BT 262.005 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 267.426 663.247 Td /F1 9.8 Tf [(Vincent Mouly)] TJ ET 0.271 0.267 0.267 rg BT 328.656 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 334.077 663.247 Td /F1 9.8 Tf [(Simone Spuler)] TJ ET 0.271 0.267 0.267 rg BT 398.017 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 403.438 663.247 Td /F1 9.8 Tf [(Ute Zacharias)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Philippi S, Bigot A, Marg A, Mouly V, Spuler S, Zacharias U. Dysferlin-deficient immortalized human myoblasts and myotubes )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(as a useful tool to study dysferlinopathy. PLOS Currents Muscular Dystrophy. 2012 Feb 28 . Edition 1. doi: )] TJ ET BT 26.250 627.533 Td /F1 9.8 Tf [(10.1371/currents.RRN1298.)] TJ ET q 15.000 22.378 577.500 602.774 re W n 0.271 0.267 0.267 rg BT 26.250 598.430 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(Dysferlin gene mutations causing LGMD2B are associated with defects in muscle membrane repair. Four stable cell lines have )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(been established from primary human dysferlin-deficient myoblasts harbouring different mutations in the dysferlin gene. We )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(have compared immortalized human myoblasts and myotubes carrying disease-causing mutations in dysferlin to their wild-type )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(counterparts. Fusion of myoblasts into myotubes and expression of muscle-specific differentiation markers were investigated )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(with special emphasis on dysferlin protein expression, subcellular localization and function in membrane repair. We found that )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(the immortalized myoblasts and myotubes were virtually indistinguishable from their parental cell line for all of the criteria we )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(investigated. They therefore will provide a very useful tool to further investigate dysferlin function and pathophysiology as well )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(as to test therapeutic strategies at the cellular level.)] TJ ET BT 26.250 458.540 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 438.586 Td /F1 9.8 Tf [(The Deutsche Forschungsgemeinschaft funded the study through Myograd \(GK1631\) and the clinical research group KFO192 )] TJ ET BT 26.250 426.681 Td /F1 9.8 Tf [(Skeletal muscle growth regulation and dysregulation. The Jain Foundation and the Association Franaise contre les )] TJ ET BT 26.250 414.777 Td /F1 9.8 Tf [(Myopathies \(AFM\) also supported this investigation.)] TJ ET BT 26.250 385.674 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(Muscular dystrophies comprise clinically and genetically heterogeneous disorders characterized by progressive weakness and )] TJ ET BT 26.250 353.815 Td /F1 9.8 Tf [(wasting of the skeletal muscle accompanied by an increase in muscle connective tissue)] TJ ET 0.267 0.267 0.267 rg BT 403.945 353.815 Td /F1 9.8 Tf [( [1])] TJ ET 0.271 0.267 0.267 rg BT 417.498 353.815 Td /F1 9.8 Tf [( Dysferlin gene mutations cause limb )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(girdle muscular dystrophy 2B \(LGMD2B\) and Miyoshi myopathy, allelic autosomal recessive diseases characterized by limb )] TJ ET BT 26.250 330.006 Td /F1 9.8 Tf [(girdle or distal weakness of early adult onset )] TJ ET 0.267 0.267 0.267 rg BT 220.792 330.006 Td /F1 9.8 Tf [([2] [3])] TJ ET 0.271 0.267 0.267 rg BT 245.186 330.006 Td /F1 9.8 Tf [( . Dysferlin \(MIM*603009\) is a 230kDa transmembrane protein comprising )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(calcium binding C2 domains that is highly expressed in skeletal muscle )] TJ ET 0.267 0.267 0.267 rg BT 335.111 318.101 Td /F1 9.8 Tf [([4] [5] )] TJ ET 0.271 0.267 0.267 rg BT 362.216 318.101 Td /F1 9.8 Tf [(. Dysferlin localizes to the sarcolemma and is )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(involved in membrane repair, membrane trafficking and muscle regeneration )] TJ ET 0.267 0.267 0.267 rg BT 358.413 306.196 Td /F1 9.8 Tf [([6] [7] [8])] TJ ET 0.271 0.267 0.267 rg BT 396.360 306.196 Td /F1 9.8 Tf [( . Various mutations associated with )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(LGMD2B have been identified in dysferlin. These mutations lead either to a reduced expression of dysferlin at the sarcolemma, )] TJ ET BT 26.250 282.387 Td /F1 9.8 Tf [(an intracelluar accumulation of dysferlin, the formation of amyloid-like deposits or to the complete absence of dysferlin protein )] TJ ET BT 26.250 270.482 Td /F1 9.8 Tf [(finally resulting in impaired muscle membrane repair )] TJ ET 0.267 0.267 0.267 rg BT 254.351 270.482 Td /F1 9.8 Tf [([9] [10] [11])] TJ ET 0.271 0.267 0.267 rg BT 303.140 270.482 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 251.077 Td /F1 9.8 Tf [(The access to primary human myoblasts from biopsies of patients with disease-causing dysferlin mutations is limited. Due to )] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(excessive fibrosis, these muscle biopsies often contain only very few myogenic cells and are highly intermingled with connective )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(tissue cells like fibroblasts and adipocytes. Additionally, primary human myoblasts in culture show a limited proliferative potential )] TJ ET BT 26.250 215.363 Td /F1 9.8 Tf [(and undergo changes that are linked to replicative senescence)] TJ ET 0.267 0.267 0.267 rg BT 296.686 215.363 Td /F1 9.8 Tf [( [12])] TJ ET 0.271 0.267 0.267 rg BT 315.659 215.363 Td /F1 9.8 Tf [( . To circumvent these limitations immortalized human )] TJ ET BT 26.250 203.458 Td /F1 9.8 Tf [(myoblast lines were generated by retroviral transduction of primary human myoblasts harbouring different disease-causing )] TJ ET BT 26.250 191.553 Td /F1 9.8 Tf [(mutations with telomerase \(hTERT\) and cyclin-dependent kinase 4 \(CDK-4\). The expression of hTERT overcomes the )] TJ ET BT 26.250 179.649 Td /F1 9.8 Tf [(progressive erosion of telomeres occuring due to cell division and the overexpression of CDK-4 blocks the induction of the p16-)] TJ ET BT 26.250 167.744 Td /F1 9.8 Tf [(mediated cellular stress-pathway )] TJ ET 0.267 0.267 0.267 rg BT 170.921 167.744 Td /F1 9.8 Tf [([13])] TJ ET 0.271 0.267 0.267 rg BT 187.184 167.744 Td /F1 9.8 Tf [( . After their immortalization these cell lines show a prolonged proliferation and )] TJ ET BT 26.250 155.839 Td /F1 9.8 Tf [(differentiation capacity compared to primary human myoblasts )] TJ ET BT 297.183 155.839 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 325.897 155.839 Td /F1 9.8 Tf [( and they can be transplanted into regenerating muscle )] TJ ET BT 26.250 143.934 Td /F5 9.8 Tf [(in vivo )] TJ ET 0.267 0.267 0.267 rg BT 56.592 143.934 Td /F1 9.8 Tf [([13])] TJ ET 0.271 0.267 0.267 rg BT 72.855 143.934 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 124.530 Td /F1 9.8 Tf [(Here we describe a detailed analysis of immortalized human myoblast lines harbouring different dysferlin mutations. These cell )] TJ ET BT 26.250 112.625 Td /F1 9.8 Tf [(lines maintain the characteristics of primary dysferlin-deficient human cell strains with respect to myogenic differentiation, )] TJ ET BT 26.250 100.720 Td /F1 9.8 Tf [(dysferlin expression and membrane repair and represent a useful tool to further investigate all aspects of dysferlin function and )] TJ ET BT 26.250 88.815 Td /F1 9.8 Tf [(dysfunction.)] TJ ET BT 26.250 52.213 Td /F4 12.0 Tf [(Material and Methods)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(1)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Muscular Dystrophy)] TJ ET Q endstream endobj 8 0 obj << /Type /Font /Subtype /Type1 /Name /F1 /BaseFont /Helvetica /Encoding /WinAnsiEncoding >> endobj 9 0 obj << /Type /Font /Subtype /Type1 /Name /F2 /BaseFont /Times-Bold /Encoding /WinAnsiEncoding >> endobj 10 0 obj << /Type /Font /Subtype /Type1 /Name /F3 /BaseFont /Times-Italic /Encoding /WinAnsiEncoding >> endobj 11 0 obj << /Type /Font /Subtype /Type1 /Name /F4 /BaseFont /Helvetica-Bold /Encoding /WinAnsiEncoding >> endobj 12 0 obj << /Type /Font /Subtype /Type1 /Name /F5 /BaseFont /Helvetica-Oblique /Encoding /WinAnsiEncoding >> endobj 13 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 1 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceGray /BitsPerComponent 8 /Length 144>> stream x1 0 'ݲ؎"e{dzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAtlM0\ endstream endobj 14 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /SMask 13 0 R /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 3 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceRGB /BitsPerComponent 8 /Length 4099>> stream xݙ,`qG8`Gv#;3dUggzB R4A0 0} 0 0ŝa0 ym۶3danangl6;ʓa{; 0(Mӯ_ !Id2LOFQu]׵"mN$I$Nܯdw0̯Y=if\.7_`ZŦrysV 0'֞=t<0 `qlwqK۶aESY& C%p8( Eї/_4'r9ooor( X:ƊD///7 â(EUUё9VAeYoooEjy*7FhvTU%rY.AumC c`,rkN'ϻ ðklV4#)MȲ ,KwEahтN'㵨(B1166YDQDy*%QA 4^r(|*a~[.#Oay4eY ! k PV\EQɘ?}Ⱥc;*q7j ʲeߣTΈ BYYzm=c<&jy!EQШyR@ = Ȋup02ʁRe4 F wa0.UU ծgt^{}GܻH40 ޝȸ B t]aw/GIGHK=O;)% :3iÿ,W x~~ -X.H#g| fh,MAf&sBa8ݧ~Շ8n 5߻ |6᚞;fi! p0$ [=\]DUe}y2 n :niuQ;yt( : deF]U(s ?Ɵ |J׆τjrã'PU'k#8;Gog?aX{4_*Ou]Xӭ8*/F>le MmݡAa Y.1!9 |xosG(c59u6w q7l+|F ah%Pa;N(H?yTgjrL(ήlf4FOJYe#Ƚ`jʲLӔODƔ[Y)O**q7zROV- wM\+aXɘQuԷn˻:}(Ċ]F͟Rqт*)7~*'@ܯx ÝG Y&cj?usJ9zaoHbTSߘ+ eB eu6o6%U4xXX 0:}3ZaeYeQavS]sf+~ "&8]/Rjڑ+a~c[|̊"c8 "rۡۨX4׊QG] c?,pqʚ1UUs;NM(G#Y䯠j>Sd 0\|E5/f:B}kݘ^j`K%gP3<: 06؉m|޶-֧ vpn \?.3+ނhV6I^0̏WiE"hݸx<;Z=9<tgڶe}gw=:$ S= "0 ,C{0eXl6{v툢ȧ|# &;hv^q-=!?n!}>*s" !4]@4BR)iީRs)q=TǘEt9vkl6*!څ~ZGڶU`˥޶l6C$I?>WIıgl63L5. 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[ 56.5920 143.0326 72.8550 152.9532 ] >> endobj 116 0 obj << /Type /Action >> endobj 117 0 obj << /Type /Page /Parent 3 0 R /Annots [ 119 0 R 121 0 R 123 0 R 125 0 R 127 0 R 129 0 R ] /Contents 118 0 R >> endobj 118 0 obj << /Length 24592 >> stream 0.271 0.267 0.267 rg q 15.000 39.610 577.500 737.390 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Patients)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(The Charit internal review board approved the study and written informed consent was obtained from all patients. Skeletal )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(muscle biopsies \(M. vastus lateralis\) were obtained from four patients with dysferlinopathy and four healthy controls. Patients )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(with LGMD2B were affected by the following mutations in dysferlin: homozygous c.4022T>C \(DYSF1\), compound heterozygous )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(c.855+1delG/c.895G>A \(DYSF2\), compound heterozygous c.1448C>A/c.*107T>A \(DYSF3\) and homozygous c.2810+2T>A )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(\(DYSF4\) \(see Table1\) resulting either in complete loss or intracellular aggregation of dysferlin. At the time of biopsies taken )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(patients were 57, 37, 36 and 25 years old, respectively.)] TJ ET BT 26.250 669.143 Td /F4 9.8 Tf [(Purification and differentiation of primary human myoblasts)] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(Primary myoblasts were isolated by protease digestion from fresh muscle biopsies and expanded at 37 )] TJ ET BT 472.225 653.626 Td /F1 8.7 Tf [(o)] TJ ET BT 477.043 649.738 Td /F1 9.8 Tf [( C in humidified )] TJ ET BT 26.250 637.833 Td /F1 9.8 Tf [(atmosphere at 5% CO )] TJ ET BT 125.417 635.769 Td /F1 8.7 Tf [(2)] TJ ET BT 130.236 637.833 Td /F1 9.8 Tf [( in skeletal muscle growth medium \(PromoCell, Heidelberg, Germany\) supplemented with 10% FCS, )] TJ ET BT 26.250 625.929 Td /F1 9.8 Tf [(glutamine \(3mM\) and gentamycin \(40g/ml\) \(Gibco, Paisely, UK\). All cultures were enriched in myoblasts by immuno-magnetic )] TJ ET BT 26.250 614.024 Td /F1 9.8 Tf [(cell sorting using anti-CD56/NCAM antibody coated magnetic beads \(Miltenyi Biotech, Bergisch Gladbach, Germany\). Purity of )] TJ ET BT 26.250 602.119 Td /F1 9.8 Tf [(the myoblast preparation was verified by staining with an anti-desmin antibody \(DAKO\) revealing more than 95% desmin-)] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(positive cells. Differentiation of myoblasts into myotubes was initiated at approximately 90% confluence by cultivation in )] TJ ET BT 26.250 578.310 Td /F1 9.8 Tf [(differentiation medium \(DMEM, 2% horse serum\) for 7 days.)] TJ ET BT 26.250 558.905 Td /F4 9.8 Tf [(Immortalization of primary human myoblasts and their differentiation into myotubes)] TJ ET BT 26.250 539.500 Td /F1 9.8 Tf [(Primary human dysferlin-deficient and control myoblast lines were transduced with pBABE retroviral vectors carrying Cdk4 and )] TJ ET BT 26.250 527.595 Td /F1 9.8 Tf [(hTERT. Puromycin and neomycin were used as selection markers, respectively and isolation of individual myogenic clones was )] TJ ET BT 26.250 515.691 Td /F1 9.8 Tf [(carried out as described by Mamchaoui et al. )] TJ ET 0.267 0.267 0.267 rg BT 222.956 515.691 Td /F1 9.8 Tf [([13])] TJ ET 0.271 0.267 0.267 rg BT 239.219 515.691 Td /F1 9.8 Tf [( . The immortalized dysferlin-deficient and control human myoblast lines were )] TJ ET BT 26.250 503.786 Td /F1 9.8 Tf [(cultured in growth medium consisting of 1 vol 199 Medium \(Invitrogen, Carlsbad, CA\)/4 vol DMEM \(Invitrogen\) supplemented )] TJ ET BT 26.250 491.881 Td /F1 9.8 Tf [(with 20% foetal calf serum \(Invitrogen\), 2.5 ng/ml HGF \(Invitrogen\), 0.1 M Dexamethasone \(Sigma-Aldrich, St. Louis, MO\) and )] TJ ET BT 26.250 479.976 Td /F1 9.8 Tf [(50g/ml Gentamycin \(Invitrogen\). Differentiation into myotubes was initiated at approximately 90% confluence by cultivation in )] TJ ET BT 26.250 468.072 Td /F1 9.8 Tf [(differentiation medium \(DMEM, 2% horse serum\) for 7 days)] TJ ET BT 26.250 448.667 Td /F4 9.8 Tf [(Immunocytochemistry)] TJ ET BT 26.250 429.262 Td /F1 9.8 Tf [(Myotubes were fixed for 10 min with 4% formaldehyde, permeabilized for 15 min with 0.2%Triton X100 and blocked with 1% )] TJ ET BT 26.250 417.357 Td /F1 9.8 Tf [(BSA diluted in PBS. The following primary antibodies were used for immunostaining: mouse monoclonal antibodies to MHCs )] TJ ET BT 26.250 405.453 Td /F1 9.8 Tf [(and dysferlin HAMLET \(both from Novocastra, Newcastle upon Tyne, UK\), desmin \(Dako\), caveolin3 \(Santa Cruz )] TJ ET BT 26.250 393.548 Td /F1 9.8 Tf [(Biotechnology\), MHCf and a-actinin \(both from Sigma-Aldrich\) in combination with secondary Alexa 488- or Alexa 568-)] TJ ET BT 26.250 381.643 Td /F1 9.8 Tf [(conjugated anti-mouse IgG antibodies \(Invitrogen\). Nuclei were counterstained with Hoechst 33258. Images were collected )] TJ ET BT 26.250 369.738 Td /F1 9.8 Tf [(using a Leica DMI6000 fluorescence microscope or with a Zeiss-LSM 510 META confocal microscope)] TJ ET BT 26.250 350.334 Td /F4 9.8 Tf [(SDS-PAGE and Western blot analysis)] TJ ET BT 26.250 330.929 Td /F1 9.8 Tf [(Myotubes were lysed and homogenized in protein extraction buffer \(50mM Tris pH 7.4, 150mM NaCl, 0.5% Triton X-100, 0.5% )] TJ ET BT 26.250 319.024 Td /F1 9.8 Tf [(Na-deoxycholate,1mM EDTA, 50mM NaF, 1mM Na-vanadate including protease inhibitors [Pierce]\). Total protein extracts )] TJ ET BT 26.250 307.119 Td /F1 9.8 Tf [(\(15g\) were separated on 10% SDS PAGE and transferred onto nitrocellulose membranes. Membranes were blocked in 5% )] TJ ET BT 26.250 295.215 Td /F1 9.8 Tf [(skimmed milk in TBS-tween 20 \(0.05%\) and probed with one of the following antibodies: mouse monoclonal antibodies to MHC )] TJ ET BT 26.250 283.310 Td /F1 9.8 Tf [(\(MF20; DSHB, Iowa City, IA,USA\), desmin \(Dako\), dysferlin \(HAMLET, Novocastra, Newcastle upon Tyne\), a-tubulin \(Sigma-)] TJ ET BT 26.250 271.405 Td /F1 9.8 Tf [(Aldrich\), caveolin3 \(Santa Cruz Biotechnology\) and a rabbit polyclonal antibody against dysferlin )] TJ ET 0.267 0.267 0.267 rg BT 443.511 271.405 Td /F1 9.8 Tf [([10] )] TJ ET 0.271 0.267 0.267 rg BT 462.485 271.405 Td /F1 9.8 Tf [(in combination with )] TJ ET BT 26.250 259.500 Td /F1 9.8 Tf [(secondary goat anti-mouse IgG or goat anti-rabbit IgG IRDye 800-conjugated antibodies \(LI-CORE, Lincoln, Nebraska USA\). )] TJ ET BT 26.250 247.596 Td /F1 9.8 Tf [(Immunoblots were visualized using the Odyssey infrared imaging system \(LI-CORE\).)] TJ ET BT 26.250 228.191 Td /F4 9.8 Tf [(Laser mediated membrane wounding)] TJ ET BT 26.250 208.786 Td /F1 9.8 Tf [(Shortly before performing the membrane wounding myotubes were washed once in Tyrode solution \(140 mM NaCl, 5 mM KCl, )] TJ ET BT 26.250 196.881 Td /F1 9.8 Tf [(2 mM MgCl)] TJ ET BT 76.082 194.817 Td /F1 8.7 Tf [(2)] TJ ET BT 80.901 196.881 Td /F1 9.8 Tf [( , 2.5 mM CaCl)] TJ ET BT 145.914 194.817 Td /F1 8.7 Tf [(2)] TJ ET BT 150.733 196.881 Td /F1 9.8 Tf [( and 10 mM HEPES, pH 7.2\). The wounding experiment was performed in Tyrode solution )] TJ ET BT 26.250 184.977 Td /F1 9.8 Tf [(supplemented with the FM1-43 dye \(2.5M; Molecular Probes, Invitrogen, Paisley, UK\). Myotubes were wounded by the )] TJ ET BT 26.250 173.072 Td /F1 9.8 Tf [(irradiation of a 2.5 x 2.5 ?m surface area for 58 s at 50% of the laser power \(30mW argon-laser\) employing a Zeiss-LSM 510 )] TJ ET BT 26.250 161.167 Td /F1 9.8 Tf [(META confocal microscope. Images were taken with a 63x oil immersion objective every 20s for 280s after wounding and were )] TJ ET BT 26.250 149.262 Td /F1 9.8 Tf [(processed using the Zeiss LSM Image Browser software. Changes of fluorescence intensity were calculated using ImageJ.)] TJ ET BT 26.250 112.660 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 92.706 Td /F4 9.8 Tf [(Generation of dysferlin-deficient immortalized human myoblast lines)] TJ ET BT 26.250 73.301 Td /F1 9.8 Tf [(Four stable cell lines were established from primary human dysferlin-deficient myoblasts harbouring different mutations in )] TJ ET BT 26.250 61.396 Td /F1 9.8 Tf [(dysferlin resulting in either the intracellular aggregation of dysferlin \(DYSF1 and DYSF2\) or the complete absence of dysferlin )] TJ ET BT 26.250 49.491 Td /F1 9.8 Tf [(protein \(DYSF3 and DYSF4\) as indicated in Table1.)] TJ ET Q q 15.000 39.610 577.500 737.390 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Patients)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(The Charit internal review board approved the study and written informed consent was obtained from all patients. Skeletal )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(muscle biopsies \(M. vastus lateralis\) were obtained from four patients with dysferlinopathy and four healthy controls. Patients )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(with LGMD2B were affected by the following mutations in dysferlin: homozygous c.4022T>C \(DYSF1\), compound heterozygous )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(c.855+1delG/c.895G>A \(DYSF2\), compound heterozygous c.1448C>A/c.*107T>A \(DYSF3\) and homozygous c.2810+2T>A )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(\(DYSF4\) \(see Table1\) resulting either in complete loss or intracellular aggregation of dysferlin. At the time of biopsies taken )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(patients were 57, 37, 36 and 25 years old, respectively.)] TJ ET BT 26.250 669.143 Td /F4 9.8 Tf [(Purification and differentiation of primary human myoblasts)] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(Primary myoblasts were isolated by protease digestion from fresh muscle biopsies and expanded at 37 )] TJ ET BT 472.225 653.626 Td /F1 8.7 Tf [(o)] TJ ET BT 477.043 649.738 Td /F1 9.8 Tf [( C in humidified )] TJ ET BT 26.250 637.833 Td /F1 9.8 Tf [(atmosphere at 5% CO )] TJ ET BT 125.417 635.769 Td /F1 8.7 Tf [(2)] TJ ET BT 130.236 637.833 Td /F1 9.8 Tf [( in skeletal muscle growth medium \(PromoCell, Heidelberg, Germany\) supplemented with 10% FCS, )] TJ ET BT 26.250 625.929 Td /F1 9.8 Tf [(glutamine \(3mM\) and gentamycin \(40g/ml\) \(Gibco, Paisely, UK\). All cultures were enriched in myoblasts by immuno-magnetic )] TJ ET BT 26.250 614.024 Td /F1 9.8 Tf [(cell sorting using anti-CD56/NCAM antibody coated magnetic beads \(Miltenyi Biotech, Bergisch Gladbach, Germany\). Purity of )] TJ ET BT 26.250 602.119 Td /F1 9.8 Tf [(the myoblast preparation was verified by staining with an anti-desmin antibody \(DAKO\) revealing more than 95% desmin-)] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(positive cells. Differentiation of myoblasts into myotubes was initiated at approximately 90% confluence by cultivation in )] TJ ET BT 26.250 578.310 Td /F1 9.8 Tf [(differentiation medium \(DMEM, 2% horse serum\) for 7 days.)] TJ ET BT 26.250 558.905 Td /F4 9.8 Tf [(Immortalization of primary human myoblasts and their differentiation into myotubes)] TJ ET BT 26.250 539.500 Td /F1 9.8 Tf [(Primary human dysferlin-deficient and control myoblast lines were transduced with pBABE retroviral vectors carrying Cdk4 and )] TJ ET BT 26.250 527.595 Td /F1 9.8 Tf [(hTERT. Puromycin and neomycin were used as selection markers, respectively and isolation of individual myogenic clones was )] TJ ET BT 26.250 515.691 Td /F1 9.8 Tf [(carried out as described by Mamchaoui et al. )] TJ ET 0.267 0.267 0.267 rg BT 222.956 515.691 Td /F1 9.8 Tf [([13])] TJ ET 0.271 0.267 0.267 rg BT 239.219 515.691 Td /F1 9.8 Tf [( . The immortalized dysferlin-deficient and control human myoblast lines were )] TJ ET BT 26.250 503.786 Td /F1 9.8 Tf [(cultured in growth medium consisting of 1 vol 199 Medium \(Invitrogen, Carlsbad, CA\)/4 vol DMEM \(Invitrogen\) supplemented )] TJ ET BT 26.250 491.881 Td /F1 9.8 Tf [(with 20% foetal calf serum \(Invitrogen\), 2.5 ng/ml HGF \(Invitrogen\), 0.1 M Dexamethasone \(Sigma-Aldrich, St. Louis, MO\) and )] TJ ET BT 26.250 479.976 Td /F1 9.8 Tf [(50g/ml Gentamycin \(Invitrogen\). Differentiation into myotubes was initiated at approximately 90% confluence by cultivation in )] TJ ET BT 26.250 468.072 Td /F1 9.8 Tf [(differentiation medium \(DMEM, 2% horse serum\) for 7 days)] TJ ET BT 26.250 448.667 Td /F4 9.8 Tf [(Immunocytochemistry)] TJ ET BT 26.250 429.262 Td /F1 9.8 Tf [(Myotubes were fixed for 10 min with 4% formaldehyde, permeabilized for 15 min with 0.2%Triton X100 and blocked with 1% )] TJ ET BT 26.250 417.357 Td /F1 9.8 Tf [(BSA diluted in PBS. The following primary antibodies were used for immunostaining: mouse monoclonal antibodies to MHCs )] TJ ET BT 26.250 405.453 Td /F1 9.8 Tf [(and dysferlin HAMLET \(both from Novocastra, Newcastle upon Tyne, UK\), desmin \(Dako\), caveolin3 \(Santa Cruz )] TJ ET BT 26.250 393.548 Td /F1 9.8 Tf [(Biotechnology\), MHCf and a-actinin \(both from Sigma-Aldrich\) in combination with secondary Alexa 488- or Alexa 568-)] TJ ET BT 26.250 381.643 Td /F1 9.8 Tf [(conjugated anti-mouse IgG antibodies \(Invitrogen\). Nuclei were counterstained with Hoechst 33258. Images were collected )] TJ ET BT 26.250 369.738 Td /F1 9.8 Tf [(using a Leica DMI6000 fluorescence microscope or with a Zeiss-LSM 510 META confocal microscope)] TJ ET BT 26.250 350.334 Td /F4 9.8 Tf [(SDS-PAGE and Western blot analysis)] TJ ET BT 26.250 330.929 Td /F1 9.8 Tf [(Myotubes were lysed and homogenized in protein extraction buffer \(50mM Tris pH 7.4, 150mM NaCl, 0.5% Triton X-100, 0.5% )] TJ ET BT 26.250 319.024 Td /F1 9.8 Tf [(Na-deoxycholate,1mM EDTA, 50mM NaF, 1mM Na-vanadate including protease inhibitors [Pierce]\). Total protein extracts )] TJ ET BT 26.250 307.119 Td /F1 9.8 Tf [(\(15g\) were separated on 10% SDS PAGE and transferred onto nitrocellulose membranes. Membranes were blocked in 5% )] TJ ET BT 26.250 295.215 Td /F1 9.8 Tf [(skimmed milk in TBS-tween 20 \(0.05%\) and probed with one of the following antibodies: mouse monoclonal antibodies to MHC )] TJ ET BT 26.250 283.310 Td /F1 9.8 Tf [(\(MF20; DSHB, Iowa City, IA,USA\), desmin \(Dako\), dysferlin \(HAMLET, Novocastra, Newcastle upon Tyne\), a-tubulin \(Sigma-)] TJ ET BT 26.250 271.405 Td /F1 9.8 Tf [(Aldrich\), caveolin3 \(Santa Cruz Biotechnology\) and a rabbit polyclonal antibody against dysferlin )] TJ ET 0.267 0.267 0.267 rg BT 443.511 271.405 Td /F1 9.8 Tf [([10] )] TJ ET 0.271 0.267 0.267 rg BT 462.485 271.405 Td /F1 9.8 Tf [(in combination with )] TJ ET BT 26.250 259.500 Td /F1 9.8 Tf [(secondary goat anti-mouse IgG or goat anti-rabbit IgG IRDye 800-conjugated antibodies \(LI-CORE, Lincoln, Nebraska USA\). )] TJ ET BT 26.250 247.596 Td /F1 9.8 Tf [(Immunoblots were visualized using the Odyssey infrared imaging system \(LI-CORE\).)] TJ ET BT 26.250 228.191 Td /F4 9.8 Tf [(Laser mediated membrane wounding)] TJ ET BT 26.250 208.786 Td /F1 9.8 Tf [(Shortly before performing the membrane wounding myotubes were washed once in Tyrode solution \(140 mM NaCl, 5 mM KCl, )] TJ ET BT 26.250 196.881 Td /F1 9.8 Tf [(2 mM MgCl)] TJ ET BT 76.082 194.817 Td /F1 8.7 Tf [(2)] TJ ET BT 80.901 196.881 Td /F1 9.8 Tf [( , 2.5 mM CaCl)] TJ ET BT 145.914 194.817 Td /F1 8.7 Tf [(2)] TJ ET BT 150.733 196.881 Td /F1 9.8 Tf [( and 10 mM HEPES, pH 7.2\). The wounding experiment was performed in Tyrode solution )] TJ ET BT 26.250 184.977 Td /F1 9.8 Tf [(supplemented with the FM1-43 dye \(2.5M; Molecular Probes, Invitrogen, Paisley, UK\). Myotubes were wounded by the )] TJ ET BT 26.250 173.072 Td /F1 9.8 Tf [(irradiation of a 2.5 x 2.5 ?m surface area for 58 s at 50% of the laser power \(30mW argon-laser\) employing a Zeiss-LSM 510 )] TJ ET BT 26.250 161.167 Td /F1 9.8 Tf [(META confocal microscope. Images were taken with a 63x oil immersion objective every 20s for 280s after wounding and were )] TJ ET BT 26.250 149.262 Td /F1 9.8 Tf [(processed using the Zeiss LSM Image Browser software. Changes of fluorescence intensity were calculated using ImageJ.)] TJ ET BT 26.250 112.660 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 92.706 Td /F4 9.8 Tf [(Generation of dysferlin-deficient immortalized human myoblast lines)] TJ ET BT 26.250 73.301 Td /F1 9.8 Tf [(Four stable cell lines were established from primary human dysferlin-deficient myoblasts harbouring different mutations in )] TJ ET BT 26.250 61.396 Td /F1 9.8 Tf [(dysferlin resulting in either the intracellular aggregation of dysferlin \(DYSF1 and DYSF2\) or the complete absence of dysferlin )] TJ ET BT 26.250 49.491 Td /F1 9.8 Tf [(protein \(DYSF3 and DYSF4\) as indicated in Table1.)] TJ ET Q q 15.000 39.610 577.500 737.390 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Patients)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(The Charit internal review board approved the study and written informed consent was obtained from all patients. Skeletal )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(muscle biopsies \(M. vastus lateralis\) were obtained from four patients with dysferlinopathy and four healthy controls. Patients )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(with LGMD2B were affected by the following mutations in dysferlin: homozygous c.4022T>C \(DYSF1\), compound heterozygous )] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(c.855+1delG/c.895G>A \(DYSF2\), compound heterozygous c.1448C>A/c.*107T>A \(DYSF3\) and homozygous c.2810+2T>A )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(\(DYSF4\) \(see Table1\) resulting either in complete loss or intracellular aggregation of dysferlin. At the time of biopsies taken )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(patients were 57, 37, 36 and 25 years old, respectively.)] TJ ET BT 26.250 669.143 Td /F4 9.8 Tf [(Purification and differentiation of primary human myoblasts)] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(Primary myoblasts were isolated by protease digestion from fresh muscle biopsies and expanded at 37 )] TJ ET BT 472.225 653.626 Td /F1 8.7 Tf [(o)] TJ ET BT 477.043 649.738 Td /F1 9.8 Tf [( C in humidified )] TJ ET BT 26.250 637.833 Td /F1 9.8 Tf [(atmosphere at 5% CO )] TJ ET BT 125.417 635.769 Td /F1 8.7 Tf [(2)] TJ ET BT 130.236 637.833 Td /F1 9.8 Tf [( in skeletal muscle growth medium \(PromoCell, Heidelberg, Germany\) supplemented with 10% FCS, )] TJ ET BT 26.250 625.929 Td /F1 9.8 Tf [(glutamine \(3mM\) and gentamycin \(40g/ml\) \(Gibco, Paisely, UK\). All cultures were enriched in myoblasts by immuno-magnetic )] TJ ET BT 26.250 614.024 Td /F1 9.8 Tf [(cell sorting using anti-CD56/NCAM antibody coated magnetic beads \(Miltenyi Biotech, Bergisch Gladbach, Germany\). Purity of )] TJ ET BT 26.250 602.119 Td /F1 9.8 Tf [(the myoblast preparation was verified by staining with an anti-desmin antibody \(DAKO\) revealing more than 95% desmin-)] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(positive cells. Differentiation of myoblasts into myotubes was initiated at approximately 90% confluence by cultivation in )] TJ ET BT 26.250 578.310 Td /F1 9.8 Tf [(differentiation medium \(DMEM, 2% horse serum\) for 7 days.)] TJ ET BT 26.250 558.905 Td /F4 9.8 Tf [(Immortalization of primary human myoblasts and their differentiation into myotubes)] TJ ET BT 26.250 539.500 Td /F1 9.8 Tf [(Primary human dysferlin-deficient and control myoblast lines were transduced with pBABE retroviral vectors carrying Cdk4 and )] TJ ET BT 26.250 527.595 Td /F1 9.8 Tf [(hTERT. Puromycin and neomycin were used as selection markers, respectively and isolation of individual myogenic clones was )] TJ ET BT 26.250 515.691 Td /F1 9.8 Tf [(carried out as described by Mamchaoui et al. )] TJ ET 0.267 0.267 0.267 rg BT 222.956 515.691 Td /F1 9.8 Tf [([13])] TJ ET 0.271 0.267 0.267 rg BT 239.219 515.691 Td /F1 9.8 Tf [( . The immortalized dysferlin-deficient and control human myoblast lines were )] TJ ET BT 26.250 503.786 Td /F1 9.8 Tf [(cultured in growth medium consisting of 1 vol 199 Medium \(Invitrogen, Carlsbad, CA\)/4 vol DMEM \(Invitrogen\) supplemented )] TJ ET BT 26.250 491.881 Td /F1 9.8 Tf [(with 20% foetal calf serum \(Invitrogen\), 2.5 ng/ml HGF \(Invitrogen\), 0.1 M Dexamethasone \(Sigma-Aldrich, St. Louis, MO\) and )] TJ ET BT 26.250 479.976 Td /F1 9.8 Tf [(50g/ml Gentamycin \(Invitrogen\). Differentiation into myotubes was initiated at approximately 90% confluence by cultivation in )] TJ ET BT 26.250 468.072 Td /F1 9.8 Tf [(differentiation medium \(DMEM, 2% horse serum\) for 7 days)] TJ ET BT 26.250 448.667 Td /F4 9.8 Tf [(Immunocytochemistry)] TJ ET BT 26.250 429.262 Td /F1 9.8 Tf [(Myotubes were fixed for 10 min with 4% formaldehyde, permeabilized for 15 min with 0.2%Triton X100 and blocked with 1% )] TJ ET BT 26.250 417.357 Td /F1 9.8 Tf [(BSA diluted in PBS. The following primary antibodies were used for immunostaining: mouse monoclonal antibodies to MHCs )] TJ ET BT 26.250 405.453 Td /F1 9.8 Tf [(and dysferlin HAMLET \(both from Novocastra, Newcastle upon Tyne, UK\), desmin \(Dako\), caveolin3 \(Santa Cruz )] TJ ET BT 26.250 393.548 Td /F1 9.8 Tf [(Biotechnology\), MHCf and a-actinin \(both from Sigma-Aldrich\) in combination with secondary Alexa 488- or Alexa 568-)] TJ ET BT 26.250 381.643 Td /F1 9.8 Tf [(conjugated anti-mouse IgG antibodies \(Invitrogen\). Nuclei were counterstained with Hoechst 33258. Images were collected )] TJ ET BT 26.250 369.738 Td /F1 9.8 Tf [(using a Leica DMI6000 fluorescence microscope or with a Zeiss-LSM 510 META confocal microscope)] TJ ET BT 26.250 350.334 Td /F4 9.8 Tf [(SDS-PAGE and Western blot analysis)] TJ ET BT 26.250 330.929 Td /F1 9.8 Tf [(Myotubes were lysed and homogenized in protein extraction buffer \(50mM Tris pH 7.4, 150mM NaCl, 0.5% Triton X-100, 0.5% )] TJ ET BT 26.250 319.024 Td /F1 9.8 Tf [(Na-deoxycholate,1mM EDTA, 50mM NaF, 1mM Na-vanadate including protease inhibitors [Pierce]\). Total protein extracts )] TJ ET BT 26.250 307.119 Td /F1 9.8 Tf [(\(15g\) were separated on 10% SDS PAGE and transferred onto nitrocellulose membranes. Membranes were blocked in 5% )] TJ ET BT 26.250 295.215 Td /F1 9.8 Tf [(skimmed milk in TBS-tween 20 \(0.05%\) and probed with one of the following antibodies: mouse monoclonal antibodies to MHC )] TJ ET BT 26.250 283.310 Td /F1 9.8 Tf [(\(MF20; DSHB, Iowa City, IA,USA\), desmin \(Dako\), dysferlin \(HAMLET, Novocastra, Newcastle upon Tyne\), a-tubulin \(Sigma-)] TJ ET BT 26.250 271.405 Td /F1 9.8 Tf [(Aldrich\), caveolin3 \(Santa Cruz Biotechnology\) and a rabbit polyclonal antibody against dysferlin )] TJ ET 0.267 0.267 0.267 rg BT 443.511 271.405 Td /F1 9.8 Tf [([10] )] TJ ET 0.271 0.267 0.267 rg BT 462.485 271.405 Td /F1 9.8 Tf [(in combination with )] TJ ET BT 26.250 259.500 Td /F1 9.8 Tf [(secondary goat anti-mouse IgG or goat anti-rabbit IgG IRDye 800-conjugated antibodies \(LI-CORE, Lincoln, Nebraska USA\). )] TJ ET BT 26.250 247.596 Td /F1 9.8 Tf [(Immunoblots were visualized using the Odyssey infrared imaging system \(LI-CORE\).)] TJ ET BT 26.250 228.191 Td /F4 9.8 Tf [(Laser mediated membrane wounding)] TJ ET BT 26.250 208.786 Td /F1 9.8 Tf [(Shortly before performing the membrane wounding myotubes were washed once in Tyrode solution \(140 mM NaCl, 5 mM KCl, )] TJ ET BT 26.250 196.881 Td /F1 9.8 Tf [(2 mM MgCl)] TJ ET BT 76.082 194.817 Td /F1 8.7 Tf [(2)] TJ ET BT 80.901 196.881 Td /F1 9.8 Tf [( , 2.5 mM CaCl)] TJ ET BT 145.914 194.817 Td /F1 8.7 Tf [(2)] TJ ET BT 150.733 196.881 Td /F1 9.8 Tf [( and 10 mM HEPES, pH 7.2\). The wounding experiment was performed in Tyrode solution )] TJ ET BT 26.250 184.977 Td /F1 9.8 Tf [(supplemented with the FM1-43 dye \(2.5M; Molecular Probes, Invitrogen, Paisley, UK\). Myotubes were wounded by the )] TJ ET BT 26.250 173.072 Td /F1 9.8 Tf [(irradiation of a 2.5 x 2.5 ?m surface area for 58 s at 50% of the laser power \(30mW argon-laser\) employing a Zeiss-LSM 510 )] TJ ET BT 26.250 161.167 Td /F1 9.8 Tf [(META confocal microscope. Images were taken with a 63x oil immersion objective every 20s for 280s after wounding and were )] TJ ET BT 26.250 149.262 Td /F1 9.8 Tf [(processed using the Zeiss LSM Image Browser software. Changes of fluorescence intensity were calculated using ImageJ.)] TJ ET BT 26.250 112.660 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 92.706 Td /F4 9.8 Tf [(Generation of dysferlin-deficient immortalized human myoblast lines)] TJ ET BT 26.250 73.301 Td /F1 9.8 Tf [(Four stable cell lines were established from primary human dysferlin-deficient myoblasts harbouring different mutations in )] TJ ET BT 26.250 61.396 Td /F1 9.8 Tf [(dysferlin resulting in either the intracellular aggregation of dysferlin \(DYSF1 and DYSF2\) or the complete absence of dysferlin )] TJ ET BT 26.250 49.491 Td /F1 9.8 Tf [(protein \(DYSF3 and DYSF4\) as indicated in Table1.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(2)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Muscular Dystrophy)] TJ ET Q endstream endobj 119 0 obj << /Type /Annot /Subtype /Link /A 120 0 R /Border [0 0 0] /H /I /Rect [ 222.9563 514.7888 239.2193 524.7094 ] >> endobj 120 0 obj << /Type /Action >> endobj 121 0 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0.267 0.267 rg q 15.000 19.831 577.500 757.169 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(After cloning, all immortalized myoblast lines were 100% myogenic as shown by the expression of both desmin and )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(CD56/NCAM by immunocytochemistry, with a total absence of connective tissue cells such as fibroblasts or adipocytes.)] TJ ET BT 26.250 736.167 Td /F4 9.8 Tf [(Table1)] TJ ET BT 57.138 736.167 Td /F1 9.8 Tf [( Summary of myoblast lines harbouring different mutations in dysferlin)] TJ ET 1.000 1.000 1.000 rg 26.250 630.117 555.000 96.169 re f 0.267 0.267 0.267 rg 26.625 725.161 85.751 0.750 re f 26.625 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 715.306 Td /F1 9.8 Tf [(Myoblast line)] TJ ET 0.267 0.267 0.267 rg 111.626 725.161 50.107 0.750 re f 111.626 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 116.876 715.306 Td /F1 9.8 Tf [(Exon)] TJ ET 0.267 0.267 0.267 rg 160.983 725.161 106.162 0.750 re f 160.983 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 166.233 715.306 Td /F1 9.8 Tf [(State)] TJ ET 0.267 0.267 0.267 rg 266.395 725.161 118.255 0.750 re f 266.395 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 271.645 715.306 Td /F1 9.8 Tf [(Nucleotide change)] TJ ET 0.267 0.267 0.267 rg 383.900 725.161 104.519 0.750 re f 383.900 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 389.150 715.306 Td /F1 9.8 Tf [(mRNA change)] TJ ET 0.267 0.267 0.267 rg 487.669 725.161 93.206 0.750 re f 487.669 709.279 0.750 16.631 re f 580.125 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 492.919 715.306 Td /F1 9.8 Tf [(Protein change)] TJ ET 0.267 0.267 0.267 rg 26.625 709.279 85.751 0.750 re f 26.625 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 699.425 Td /F1 9.8 Tf [(DYSF1)] TJ ET 0.267 0.267 0.267 rg 111.626 709.279 50.107 0.750 re f 111.626 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 116.876 699.425 Td /F1 9.8 Tf [(38)] TJ ET 0.267 0.267 0.267 rg 160.983 709.279 106.162 0.750 re f 160.983 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 166.233 699.425 Td /F1 9.8 Tf [(homozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 709.279 118.255 0.750 re f 266.395 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 271.645 699.425 Td /F1 9.8 Tf [(c.4022T>C)] TJ ET 0.267 0.267 0.267 rg 383.900 709.279 104.519 0.750 re f 383.900 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 389.150 699.425 Td /F1 9.8 Tf [(r.4022u>c)] TJ ET 0.267 0.267 0.267 rg 487.669 709.279 93.206 0.750 re f 487.669 693.398 0.750 16.631 re f 580.125 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 492.919 699.425 Td /F1 9.8 Tf [(p.L1341P)] TJ ET 0.267 0.267 0.267 rg 26.625 693.398 85.751 0.750 re f 26.625 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 31.875 683.543 Td /F1 9.8 Tf [(DYSF2)] TJ ET 0.267 0.267 0.267 rg 111.626 693.398 50.107 0.750 re f 111.626 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 116.876 683.543 Td /F1 9.8 Tf [(8i)] TJ ET BT 116.876 675.912 Td /F1 9.8 Tf [(9)] TJ ET 0.267 0.267 0.267 rg 160.983 693.398 106.162 0.750 re f 160.983 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 166.233 683.543 Td /F1 9.8 Tf [(heterozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 693.398 118.255 0.750 re f 266.395 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 271.645 683.543 Td /F1 9.8 Tf [(c.855+1delG)] TJ ET BT 271.645 675.912 Td /F1 9.8 Tf [(c.895G>A)] TJ ET 0.267 0.267 0.267 rg 383.900 693.398 104.519 0.750 re f 383.900 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 389.150 683.543 Td /F1 9.8 Tf [(splice site mutation)] TJ ET BT 389.150 675.912 Td /F1 9.8 Tf [(r.895g>a)] TJ ET 0.267 0.267 0.267 rg 487.669 693.398 93.206 0.750 re f 487.669 669.886 0.750 24.262 re f 580.125 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 492.919 676.043 Td /F1 9.8 Tf [(p.G299R)] TJ ET 0.267 0.267 0.267 rg 26.625 669.886 85.751 0.750 re f 26.625 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 31.875 660.031 Td /F1 9.8 Tf [(DYSF3)] TJ ET 0.267 0.267 0.267 rg 111.626 669.886 50.107 0.750 re f 111.626 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 116.876 660.031 Td /F1 9.8 Tf [(16)] TJ ET BT 116.876 652.400 Td /F1 9.8 Tf [(55)] TJ ET 0.267 0.267 0.267 rg 160.983 669.886 106.162 0.750 re f 160.983 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 166.233 660.031 Td /F1 9.8 Tf [(heterozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 669.886 118.255 0.750 re f 266.395 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 271.645 660.031 Td /F1 9.8 Tf [(c.1448C>A)] TJ ET BT 271.645 652.400 Td /F1 9.8 Tf [(c.*107T>A)] TJ ET 0.267 0.267 0.267 rg 383.900 669.886 104.519 0.750 re f 383.900 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 389.150 660.031 Td /F1 9.8 Tf [(r.1448c>a)] TJ ET BT 389.150 652.400 Td /F1 9.8 Tf [(r.*107u>a)] TJ ET 0.267 0.267 0.267 rg 487.669 669.886 93.206 0.750 re f 487.669 646.373 0.750 24.262 re f 580.125 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 492.919 660.031 Td /F1 9.8 Tf [(p.S483X)] TJ ET 0.267 0.267 0.267 rg 26.625 646.373 85.751 0.750 re f 26.625 630.492 85.751 0.750 re f 26.625 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 636.518 Td /F1 9.8 Tf [(DYSF4)] TJ ET 0.267 0.267 0.267 rg 111.626 646.373 50.107 0.750 re f 111.626 630.492 50.107 0.750 re f 111.626 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 116.876 636.518 Td /F1 9.8 Tf [(26i)] TJ ET 0.267 0.267 0.267 rg 160.983 646.373 106.162 0.750 re f 160.983 630.492 106.162 0.750 re f 160.983 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 166.233 636.518 Td /F1 9.8 Tf [(homozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 646.373 118.255 0.750 re f 266.395 630.492 118.255 0.750 re f 266.395 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 271.645 636.518 Td /F1 9.8 Tf [(c.2810+2T>A)] TJ ET 0.267 0.267 0.267 rg 383.900 646.373 104.519 0.750 re f 383.900 630.492 104.519 0.750 re f 383.900 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 389.150 636.518 Td /F1 9.8 Tf [(splice site mutation)] TJ ET 0.267 0.267 0.267 rg 487.669 646.373 93.206 0.750 re f 487.669 630.492 93.206 0.750 re f 487.669 630.492 0.750 16.631 re f 580.125 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 26.250 583.093 Td /F1 9.8 Tf [(The reference sequence used is GenBank ID NM_003494. The nomenclature sequence uses c, r and p when referring to )] TJ ET BT 26.250 571.188 Td /F1 9.8 Tf [(cDNA, mRNA and protein, respectively, and i when referring to intronic sequence.)] TJ ET BT 26.250 551.784 Td /F4 9.8 Tf [(Comparison of dysferlin-deficient immortalized human myoblasts and myotubes to their parental cell lines)] TJ ET BT 26.250 532.379 Td /F1 9.8 Tf [(The effect of immortalization on myoblast differentiation into myotubes was analyzed by Western blott assessing the expression )] TJ ET BT 26.250 520.474 Td /F1 9.8 Tf [(of myogenic differentiation markers. All immortalized human myoblast lines showed a strong expression of desmin and a weak )] TJ ET BT 26.250 508.569 Td /F1 9.8 Tf [(expression of caveolin3 \(Fig.1B\) similar to their parent myoblast lines \(Fig.1A\). Primary and immortalized \(IM\) wild-type human )] TJ ET BT 26.250 496.665 Td /F1 9.8 Tf [(myoblasts showed a low expression of dysferlin, that was reduced in IM DYSF1 and DYSF2/IM DYSF2 and completely absent )] TJ ET BT 26.250 484.760 Td /F1 9.8 Tf [(in DYSF3/IM DYSF3 and DYSF4/IM DYSF4. We were not able to analyze primary human DYSF1 myoblasts and their )] TJ ET BT 26.250 472.855 Td /F1 9.8 Tf [(corresponding myotubes due to a massive presence of non-muscle cell types and a very poor myogenicity, further enhancing )] TJ ET BT 26.250 460.950 Td /F1 9.8 Tf [(the interest in generating immortalized clones from patients primary cultures.)] TJ ET BT 26.250 441.546 Td /F1 9.8 Tf [(After 7 days of differentiation myotube formation was associated with a strong increase in the expression of myosin heavy chain )] TJ ET BT 26.250 429.641 Td /F1 9.8 Tf [(\(MHC\) and caveolin3 in all primary and immortalized cell lines investigated \(Fig.1A and 1B\) except DYSF1 regardless of the )] TJ ET BT 26.250 417.736 Td /F1 9.8 Tf [(presence of dysferlin. Desmin was still expressed in myotubes. a-tubulin served as a loading control. Myotubes derived from )] TJ ET BT 26.250 405.831 Td /F1 9.8 Tf [(primary and immortalized wild-type myoblasts showed a strong increase in dysferlin expression with differentiation \(Fig.1A and )] TJ ET BT 26.250 393.927 Td /F1 9.8 Tf [(1B\). As expected, myotubes with disease-causing mutations in DYSF showed a strongly reduced expression \(IM DYSF1 and )] TJ ET BT 26.250 382.022 Td /F1 9.8 Tf [(DYSF2/IM DYSF2\) or a complete absence of dysferlin protein \(DYSF3/IM DYSF3 and DYSF4/IM DYSF4\))] TJ ET 0.965 0.965 0.965 rg 26.250 145.069 555.000 227.072 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 372.141 m 581.250 372.141 l 581.250 371.391 l 26.250 371.391 l f 26.250 145.069 m 581.250 145.069 l 581.250 145.819 l 26.250 145.819 l f q 225.000 0 0 92.250 35.250 270.141 cm /I3 Do Q q 35.250 156.319 537.000 107.822 re W n 0.271 0.267 0.267 rg BT 35.250 254.617 Td /F4 9.8 Tf [(Fig. 1: Comparison of immortalized human myoblasts and myotubes to their parental counterparts.)] TJ ET BT 35.250 235.247 Td /F1 9.8 Tf [(The expression of myogenic differentiation markers was analysed by Western blot in dysferlin-deficient and wild-type )] TJ ET BT 35.250 221.511 Td /F1 9.8 Tf [(human myoblasts and myotubes in primary cells \(A\) and after immortalization \(B\). A similar increase in MHC \(200kDa\) and )] TJ ET BT 35.250 207.775 Td /F1 9.8 Tf [(caveolin3 \(23kDa\) was observed in primary \(A\) and immortalized \(IM\) \(B\) cell lines after differentiation into myotubes. )] TJ ET BT 35.250 194.038 Td /F1 9.8 Tf [(Mutations in dysferlin result in a reduced expression \(IM DYSF1 and DYSF2/IM DYSF2\) or in a complete absence of )] TJ ET BT 35.250 180.302 Td /F1 9.8 Tf [(dysferlin protein \(230kDa\) \(DYSF3/IM DYSF3 and DYSF4/IM DYSF4\) in both myoblasts and myotubes. a-tubulin \(50kDa\) )] TJ ET BT 35.250 166.566 Td /F1 9.8 Tf [(has been used as a loading control.)] TJ ET Q BT 26.250 128.045 Td /F1 9.8 Tf [(In primary and immortalized wild-type myotubes and in IM DYSF1 and DYSF2/IM DYSF2 myotubes dysferlin was only )] TJ ET BT 26.250 116.140 Td /F1 9.8 Tf [(expressed as a single 230 kDa protein band as revealed by the HAMLET antibody directed against the C-terminal )] TJ ET BT 26.250 104.235 Td /F1 9.8 Tf [(juxtamembrane region of dysferlin and by a polyclonal antibody to the N-terminal part of dysferlin)] TJ ET 0.267 0.267 0.267 rg BT 442.965 104.235 Td /F1 9.8 Tf [( [10] )] TJ ET 0.271 0.267 0.267 rg BT 464.649 104.235 Td /F1 9.8 Tf [(\(data not shown\). This )] TJ ET BT 26.250 92.331 Td /F1 9.8 Tf [(suggests that dysferlin protein with disease causing-point mutations is expressed as the full-length protein.)] TJ ET BT 26.250 72.926 Td /F1 9.8 Tf [(Altogether dysferlin-deficient immortalized human myoblasts showed a differentiation pattern comparable to their untransformed )] TJ ET BT 26.250 61.021 Td /F1 9.8 Tf [(counterparts. Furthermore, the differentiation of myoblasts with disease-causing mutations in dysferlin into myotubes is similar )] TJ ET BT 26.250 49.116 Td /F1 9.8 Tf [(to wild-type myoblasts with respect to the expression of myogenic differentiation markers such as desmin, MHC and caveolin3.)] TJ ET Q q 15.000 19.831 577.500 757.169 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(After cloning, all immortalized myoblast lines were 100% myogenic as shown by the expression of both desmin and )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(CD56/NCAM by immunocytochemistry, with a total absence of connective tissue cells such as fibroblasts or adipocytes.)] TJ ET BT 26.250 736.167 Td /F4 9.8 Tf [(Table1)] TJ ET BT 57.138 736.167 Td /F1 9.8 Tf [( Summary of myoblast lines harbouring different mutations in dysferlin)] TJ ET 1.000 1.000 1.000 rg 26.250 630.117 555.000 96.169 re f 0.267 0.267 0.267 rg 26.625 725.161 85.751 0.750 re f 26.625 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 715.306 Td /F1 9.8 Tf [(Myoblast line)] TJ ET 0.267 0.267 0.267 rg 111.626 725.161 50.107 0.750 re f 111.626 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 116.876 715.306 Td /F1 9.8 Tf [(Exon)] TJ ET 0.267 0.267 0.267 rg 160.983 725.161 106.162 0.750 re f 160.983 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 166.233 715.306 Td /F1 9.8 Tf [(State)] TJ ET 0.267 0.267 0.267 rg 266.395 725.161 118.255 0.750 re f 266.395 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 271.645 715.306 Td /F1 9.8 Tf [(Nucleotide change)] TJ ET 0.267 0.267 0.267 rg 383.900 725.161 104.519 0.750 re f 383.900 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 389.150 715.306 Td /F1 9.8 Tf [(mRNA change)] TJ ET 0.267 0.267 0.267 rg 487.669 725.161 93.206 0.750 re f 487.669 709.279 0.750 16.631 re f 580.125 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 492.919 715.306 Td /F1 9.8 Tf [(Protein change)] TJ ET 0.267 0.267 0.267 rg 26.625 709.279 85.751 0.750 re f 26.625 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 699.425 Td /F1 9.8 Tf [(DYSF1)] TJ ET 0.267 0.267 0.267 rg 111.626 709.279 50.107 0.750 re f 111.626 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 116.876 699.425 Td /F1 9.8 Tf [(38)] TJ ET 0.267 0.267 0.267 rg 160.983 709.279 106.162 0.750 re f 160.983 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 166.233 699.425 Td /F1 9.8 Tf [(homozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 709.279 118.255 0.750 re f 266.395 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 271.645 699.425 Td /F1 9.8 Tf [(c.4022T>C)] TJ ET 0.267 0.267 0.267 rg 383.900 709.279 104.519 0.750 re f 383.900 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 389.150 699.425 Td /F1 9.8 Tf [(r.4022u>c)] TJ ET 0.267 0.267 0.267 rg 487.669 709.279 93.206 0.750 re f 487.669 693.398 0.750 16.631 re f 580.125 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 492.919 699.425 Td /F1 9.8 Tf [(p.L1341P)] TJ ET 0.267 0.267 0.267 rg 26.625 693.398 85.751 0.750 re f 26.625 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 31.875 683.543 Td /F1 9.8 Tf [(DYSF2)] TJ ET 0.267 0.267 0.267 rg 111.626 693.398 50.107 0.750 re f 111.626 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 116.876 683.543 Td /F1 9.8 Tf [(8i)] TJ ET BT 116.876 675.912 Td /F1 9.8 Tf [(9)] TJ ET 0.267 0.267 0.267 rg 160.983 693.398 106.162 0.750 re f 160.983 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 166.233 683.543 Td /F1 9.8 Tf [(heterozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 693.398 118.255 0.750 re f 266.395 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 271.645 683.543 Td /F1 9.8 Tf [(c.855+1delG)] TJ ET BT 271.645 675.912 Td /F1 9.8 Tf [(c.895G>A)] TJ ET 0.267 0.267 0.267 rg 383.900 693.398 104.519 0.750 re f 383.900 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 389.150 683.543 Td /F1 9.8 Tf [(splice site mutation)] TJ ET BT 389.150 675.912 Td /F1 9.8 Tf [(r.895g>a)] TJ ET 0.267 0.267 0.267 rg 487.669 693.398 93.206 0.750 re f 487.669 669.886 0.750 24.262 re f 580.125 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 492.919 676.043 Td /F1 9.8 Tf [(p.G299R)] TJ ET 0.267 0.267 0.267 rg 26.625 669.886 85.751 0.750 re f 26.625 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 31.875 660.031 Td /F1 9.8 Tf [(DYSF3)] TJ ET 0.267 0.267 0.267 rg 111.626 669.886 50.107 0.750 re f 111.626 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 116.876 660.031 Td /F1 9.8 Tf [(16)] TJ ET BT 116.876 652.400 Td /F1 9.8 Tf [(55)] TJ ET 0.267 0.267 0.267 rg 160.983 669.886 106.162 0.750 re f 160.983 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 166.233 660.031 Td /F1 9.8 Tf [(heterozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 669.886 118.255 0.750 re f 266.395 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 271.645 660.031 Td /F1 9.8 Tf [(c.1448C>A)] TJ ET BT 271.645 652.400 Td /F1 9.8 Tf [(c.*107T>A)] TJ ET 0.267 0.267 0.267 rg 383.900 669.886 104.519 0.750 re f 383.900 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 389.150 660.031 Td /F1 9.8 Tf [(r.1448c>a)] TJ ET BT 389.150 652.400 Td /F1 9.8 Tf [(r.*107u>a)] TJ ET 0.267 0.267 0.267 rg 487.669 669.886 93.206 0.750 re f 487.669 646.373 0.750 24.262 re f 580.125 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 492.919 660.031 Td /F1 9.8 Tf [(p.S483X)] TJ ET 0.267 0.267 0.267 rg 26.625 646.373 85.751 0.750 re f 26.625 630.492 85.751 0.750 re f 26.625 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 636.518 Td /F1 9.8 Tf [(DYSF4)] TJ ET 0.267 0.267 0.267 rg 111.626 646.373 50.107 0.750 re f 111.626 630.492 50.107 0.750 re f 111.626 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 116.876 636.518 Td /F1 9.8 Tf [(26i)] TJ ET 0.267 0.267 0.267 rg 160.983 646.373 106.162 0.750 re f 160.983 630.492 106.162 0.750 re f 160.983 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 166.233 636.518 Td /F1 9.8 Tf [(homozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 646.373 118.255 0.750 re f 266.395 630.492 118.255 0.750 re f 266.395 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 271.645 636.518 Td /F1 9.8 Tf [(c.2810+2T>A)] TJ ET 0.267 0.267 0.267 rg 383.900 646.373 104.519 0.750 re f 383.900 630.492 104.519 0.750 re f 383.900 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 389.150 636.518 Td /F1 9.8 Tf [(splice site mutation)] TJ ET 0.267 0.267 0.267 rg 487.669 646.373 93.206 0.750 re f 487.669 630.492 93.206 0.750 re f 487.669 630.492 0.750 16.631 re f 580.125 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 26.250 583.093 Td /F1 9.8 Tf [(The reference sequence used is GenBank ID NM_003494. The nomenclature sequence uses c, r and p when referring to )] TJ ET BT 26.250 571.188 Td /F1 9.8 Tf [(cDNA, mRNA and protein, respectively, and i when referring to intronic sequence.)] TJ ET BT 26.250 551.784 Td /F4 9.8 Tf [(Comparison of dysferlin-deficient immortalized human myoblasts and myotubes to their parental cell lines)] TJ ET BT 26.250 532.379 Td /F1 9.8 Tf [(The effect of immortalization on myoblast differentiation into myotubes was analyzed by Western blott assessing the expression )] TJ ET BT 26.250 520.474 Td /F1 9.8 Tf [(of myogenic differentiation markers. All immortalized human myoblast lines showed a strong expression of desmin and a weak )] TJ ET BT 26.250 508.569 Td /F1 9.8 Tf [(expression of caveolin3 \(Fig.1B\) similar to their parent myoblast lines \(Fig.1A\). Primary and immortalized \(IM\) wild-type human )] TJ ET BT 26.250 496.665 Td /F1 9.8 Tf [(myoblasts showed a low expression of dysferlin, that was reduced in IM DYSF1 and DYSF2/IM DYSF2 and completely absent )] TJ ET BT 26.250 484.760 Td /F1 9.8 Tf [(in DYSF3/IM DYSF3 and DYSF4/IM DYSF4. We were not able to analyze primary human DYSF1 myoblasts and their )] TJ ET BT 26.250 472.855 Td /F1 9.8 Tf [(corresponding myotubes due to a massive presence of non-muscle cell types and a very poor myogenicity, further enhancing )] TJ ET BT 26.250 460.950 Td /F1 9.8 Tf [(the interest in generating immortalized clones from patients primary cultures.)] TJ ET BT 26.250 441.546 Td /F1 9.8 Tf [(After 7 days of differentiation myotube formation was associated with a strong increase in the expression of myosin heavy chain )] TJ ET BT 26.250 429.641 Td /F1 9.8 Tf [(\(MHC\) and caveolin3 in all primary and immortalized cell lines investigated \(Fig.1A and 1B\) except DYSF1 regardless of the )] TJ ET BT 26.250 417.736 Td /F1 9.8 Tf [(presence of dysferlin. Desmin was still expressed in myotubes. a-tubulin served as a loading control. Myotubes derived from )] TJ ET BT 26.250 405.831 Td /F1 9.8 Tf [(primary and immortalized wild-type myoblasts showed a strong increase in dysferlin expression with differentiation \(Fig.1A and )] TJ ET BT 26.250 393.927 Td /F1 9.8 Tf [(1B\). As expected, myotubes with disease-causing mutations in DYSF showed a strongly reduced expression \(IM DYSF1 and )] TJ ET BT 26.250 382.022 Td /F1 9.8 Tf [(DYSF2/IM DYSF2\) or a complete absence of dysferlin protein \(DYSF3/IM DYSF3 and DYSF4/IM DYSF4\))] TJ ET 0.965 0.965 0.965 rg 26.250 145.069 555.000 227.072 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 372.141 m 581.250 372.141 l 581.250 371.391 l 26.250 371.391 l f 26.250 145.069 m 581.250 145.069 l 581.250 145.819 l 26.250 145.819 l f q 225.000 0 0 92.250 35.250 270.141 cm /I3 Do Q q 35.250 156.319 537.000 107.822 re W n 0.271 0.267 0.267 rg BT 35.250 254.617 Td /F4 9.8 Tf [(Fig. 1: Comparison of immortalized human myoblasts and myotubes to their parental counterparts.)] TJ ET BT 35.250 235.247 Td /F1 9.8 Tf [(The expression of myogenic differentiation markers was analysed by Western blot in dysferlin-deficient and wild-type )] TJ ET BT 35.250 221.511 Td /F1 9.8 Tf [(human myoblasts and myotubes in primary cells \(A\) and after immortalization \(B\). A similar increase in MHC \(200kDa\) and )] TJ ET BT 35.250 207.775 Td /F1 9.8 Tf [(caveolin3 \(23kDa\) was observed in primary \(A\) and immortalized \(IM\) \(B\) cell lines after differentiation into myotubes. )] TJ ET BT 35.250 194.038 Td /F1 9.8 Tf [(Mutations in dysferlin result in a reduced expression \(IM DYSF1 and DYSF2/IM DYSF2\) or in a complete absence of )] TJ ET BT 35.250 180.302 Td /F1 9.8 Tf [(dysferlin protein \(230kDa\) \(DYSF3/IM DYSF3 and DYSF4/IM DYSF4\) in both myoblasts and myotubes. a-tubulin \(50kDa\) )] TJ ET BT 35.250 166.566 Td /F1 9.8 Tf [(has been used as a loading control.)] TJ ET Q BT 26.250 128.045 Td /F1 9.8 Tf [(In primary and immortalized wild-type myotubes and in IM DYSF1 and DYSF2/IM DYSF2 myotubes dysferlin was only )] TJ ET BT 26.250 116.140 Td /F1 9.8 Tf [(expressed as a single 230 kDa protein band as revealed by the HAMLET antibody directed against the C-terminal )] TJ ET BT 26.250 104.235 Td /F1 9.8 Tf [(juxtamembrane region of dysferlin and by a polyclonal antibody to the N-terminal part of dysferlin)] TJ ET 0.267 0.267 0.267 rg BT 442.965 104.235 Td /F1 9.8 Tf [( [10] )] TJ ET 0.271 0.267 0.267 rg BT 464.649 104.235 Td /F1 9.8 Tf [(\(data not shown\). This )] TJ ET BT 26.250 92.331 Td /F1 9.8 Tf [(suggests that dysferlin protein with disease causing-point mutations is expressed as the full-length protein.)] TJ ET BT 26.250 72.926 Td /F1 9.8 Tf [(Altogether dysferlin-deficient immortalized human myoblasts showed a differentiation pattern comparable to their untransformed )] TJ ET BT 26.250 61.021 Td /F1 9.8 Tf [(counterparts. Furthermore, the differentiation of myoblasts with disease-causing mutations in dysferlin into myotubes is similar )] TJ ET BT 26.250 49.116 Td /F1 9.8 Tf [(to wild-type myoblasts with respect to the expression of myogenic differentiation markers such as desmin, MHC and caveolin3.)] TJ ET Q q 15.000 19.831 577.500 757.169 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(After cloning, all immortalized myoblast lines were 100% myogenic as shown by the expression of both desmin and )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(CD56/NCAM by immunocytochemistry, with a total absence of connective tissue cells such as fibroblasts or adipocytes.)] TJ ET BT 26.250 736.167 Td /F4 9.8 Tf [(Table1)] TJ ET BT 57.138 736.167 Td /F1 9.8 Tf [( Summary of myoblast lines harbouring different mutations in dysferlin)] TJ ET 1.000 1.000 1.000 rg 26.250 630.117 555.000 96.169 re f 0.267 0.267 0.267 rg 26.625 725.161 85.751 0.750 re f 26.625 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 715.306 Td /F1 9.8 Tf [(Myoblast line)] TJ ET 0.267 0.267 0.267 rg 111.626 725.161 50.107 0.750 re f 111.626 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 116.876 715.306 Td /F1 9.8 Tf [(Exon)] TJ ET 0.267 0.267 0.267 rg 160.983 725.161 106.162 0.750 re f 160.983 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 166.233 715.306 Td /F1 9.8 Tf [(State)] TJ ET 0.267 0.267 0.267 rg 266.395 725.161 118.255 0.750 re f 266.395 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 271.645 715.306 Td /F1 9.8 Tf [(Nucleotide change)] TJ ET 0.267 0.267 0.267 rg 383.900 725.161 104.519 0.750 re f 383.900 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 389.150 715.306 Td /F1 9.8 Tf [(mRNA change)] TJ ET 0.267 0.267 0.267 rg 487.669 725.161 93.206 0.750 re f 487.669 709.279 0.750 16.631 re f 580.125 709.279 0.750 16.631 re f 0.271 0.267 0.267 rg BT 492.919 715.306 Td /F1 9.8 Tf [(Protein change)] TJ ET 0.267 0.267 0.267 rg 26.625 709.279 85.751 0.750 re f 26.625 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 699.425 Td /F1 9.8 Tf [(DYSF1)] TJ ET 0.267 0.267 0.267 rg 111.626 709.279 50.107 0.750 re f 111.626 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 116.876 699.425 Td /F1 9.8 Tf [(38)] TJ ET 0.267 0.267 0.267 rg 160.983 709.279 106.162 0.750 re f 160.983 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 166.233 699.425 Td /F1 9.8 Tf [(homozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 709.279 118.255 0.750 re f 266.395 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 271.645 699.425 Td /F1 9.8 Tf [(c.4022T>C)] TJ ET 0.267 0.267 0.267 rg 383.900 709.279 104.519 0.750 re f 383.900 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 389.150 699.425 Td /F1 9.8 Tf [(r.4022u>c)] TJ ET 0.267 0.267 0.267 rg 487.669 709.279 93.206 0.750 re f 487.669 693.398 0.750 16.631 re f 580.125 693.398 0.750 16.631 re f 0.271 0.267 0.267 rg BT 492.919 699.425 Td /F1 9.8 Tf [(p.L1341P)] TJ ET 0.267 0.267 0.267 rg 26.625 693.398 85.751 0.750 re f 26.625 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 31.875 683.543 Td /F1 9.8 Tf [(DYSF2)] TJ ET 0.267 0.267 0.267 rg 111.626 693.398 50.107 0.750 re f 111.626 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 116.876 683.543 Td /F1 9.8 Tf [(8i)] TJ ET BT 116.876 675.912 Td /F1 9.8 Tf [(9)] TJ ET 0.267 0.267 0.267 rg 160.983 693.398 106.162 0.750 re f 160.983 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 166.233 683.543 Td /F1 9.8 Tf [(heterozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 693.398 118.255 0.750 re f 266.395 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 271.645 683.543 Td /F1 9.8 Tf [(c.855+1delG)] TJ ET BT 271.645 675.912 Td /F1 9.8 Tf [(c.895G>A)] TJ ET 0.267 0.267 0.267 rg 383.900 693.398 104.519 0.750 re f 383.900 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 389.150 683.543 Td /F1 9.8 Tf [(splice site mutation)] TJ ET BT 389.150 675.912 Td /F1 9.8 Tf [(r.895g>a)] TJ ET 0.267 0.267 0.267 rg 487.669 693.398 93.206 0.750 re f 487.669 669.886 0.750 24.262 re f 580.125 669.886 0.750 24.262 re f 0.271 0.267 0.267 rg BT 492.919 676.043 Td /F1 9.8 Tf [(p.G299R)] TJ ET 0.267 0.267 0.267 rg 26.625 669.886 85.751 0.750 re f 26.625 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 31.875 660.031 Td /F1 9.8 Tf [(DYSF3)] TJ ET 0.267 0.267 0.267 rg 111.626 669.886 50.107 0.750 re f 111.626 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 116.876 660.031 Td /F1 9.8 Tf [(16)] TJ ET BT 116.876 652.400 Td /F1 9.8 Tf [(55)] TJ ET 0.267 0.267 0.267 rg 160.983 669.886 106.162 0.750 re f 160.983 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 166.233 660.031 Td /F1 9.8 Tf [(heterozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 669.886 118.255 0.750 re f 266.395 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 271.645 660.031 Td /F1 9.8 Tf [(c.1448C>A)] TJ ET BT 271.645 652.400 Td /F1 9.8 Tf [(c.*107T>A)] TJ ET 0.267 0.267 0.267 rg 383.900 669.886 104.519 0.750 re f 383.900 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 389.150 660.031 Td /F1 9.8 Tf [(r.1448c>a)] TJ ET BT 389.150 652.400 Td /F1 9.8 Tf [(r.*107u>a)] TJ ET 0.267 0.267 0.267 rg 487.669 669.886 93.206 0.750 re f 487.669 646.373 0.750 24.262 re f 580.125 646.373 0.750 24.262 re f 0.271 0.267 0.267 rg BT 492.919 660.031 Td /F1 9.8 Tf [(p.S483X)] TJ ET 0.267 0.267 0.267 rg 26.625 646.373 85.751 0.750 re f 26.625 630.492 85.751 0.750 re f 26.625 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 31.875 636.518 Td /F1 9.8 Tf [(DYSF4)] TJ ET 0.267 0.267 0.267 rg 111.626 646.373 50.107 0.750 re f 111.626 630.492 50.107 0.750 re f 111.626 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 116.876 636.518 Td /F1 9.8 Tf [(26i)] TJ ET 0.267 0.267 0.267 rg 160.983 646.373 106.162 0.750 re f 160.983 630.492 106.162 0.750 re f 160.983 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 166.233 636.518 Td /F1 9.8 Tf [(homozygous)] TJ ET 0.267 0.267 0.267 rg 266.395 646.373 118.255 0.750 re f 266.395 630.492 118.255 0.750 re f 266.395 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 271.645 636.518 Td /F1 9.8 Tf [(c.2810+2T>A)] TJ ET 0.267 0.267 0.267 rg 383.900 646.373 104.519 0.750 re f 383.900 630.492 104.519 0.750 re f 383.900 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 389.150 636.518 Td /F1 9.8 Tf [(splice site mutation)] TJ ET 0.267 0.267 0.267 rg 487.669 646.373 93.206 0.750 re f 487.669 630.492 93.206 0.750 re f 487.669 630.492 0.750 16.631 re f 580.125 630.492 0.750 16.631 re f 0.271 0.267 0.267 rg BT 26.250 583.093 Td /F1 9.8 Tf [(The reference sequence used is GenBank ID NM_003494. The nomenclature sequence uses c, r and p when referring to )] TJ ET BT 26.250 571.188 Td /F1 9.8 Tf [(cDNA, mRNA and protein, respectively, and i when referring to intronic sequence.)] TJ ET BT 26.250 551.784 Td /F4 9.8 Tf [(Comparison of dysferlin-deficient immortalized human myoblasts and myotubes to their parental cell lines)] TJ ET BT 26.250 532.379 Td /F1 9.8 Tf [(The effect of immortalization on myoblast differentiation into myotubes was analyzed by Western blott assessing the expression )] TJ ET BT 26.250 520.474 Td /F1 9.8 Tf [(of myogenic differentiation markers. All immortalized human myoblast lines showed a strong expression of desmin and a weak )] TJ ET BT 26.250 508.569 Td /F1 9.8 Tf [(expression of caveolin3 \(Fig.1B\) similar to their parent myoblast lines \(Fig.1A\). Primary and immortalized \(IM\) wild-type human )] TJ ET BT 26.250 496.665 Td /F1 9.8 Tf [(myoblasts showed a low expression of dysferlin, that was reduced in IM DYSF1 and DYSF2/IM DYSF2 and completely absent )] TJ ET BT 26.250 484.760 Td /F1 9.8 Tf [(in DYSF3/IM DYSF3 and DYSF4/IM DYSF4. We were not able to analyze primary human DYSF1 myoblasts and their )] TJ ET BT 26.250 472.855 Td /F1 9.8 Tf [(corresponding myotubes due to a massive presence of non-muscle cell types and a very poor myogenicity, further enhancing )] TJ ET BT 26.250 460.950 Td /F1 9.8 Tf [(the interest in generating immortalized clones from patients primary cultures.)] TJ ET BT 26.250 441.546 Td /F1 9.8 Tf [(After 7 days of differentiation myotube formation was associated with a strong increase in the expression of myosin heavy chain )] TJ ET BT 26.250 429.641 Td /F1 9.8 Tf [(\(MHC\) and caveolin3 in all primary and immortalized cell lines investigated \(Fig.1A and 1B\) except DYSF1 regardless of the )] TJ ET BT 26.250 417.736 Td /F1 9.8 Tf [(presence of dysferlin. Desmin was still expressed in myotubes. a-tubulin served as a loading control. Myotubes derived from )] TJ ET BT 26.250 405.831 Td /F1 9.8 Tf [(primary and immortalized wild-type myoblasts showed a strong increase in dysferlin expression with differentiation \(Fig.1A and )] TJ ET BT 26.250 393.927 Td /F1 9.8 Tf [(1B\). As expected, myotubes with disease-causing mutations in DYSF showed a strongly reduced expression \(IM DYSF1 and )] TJ ET BT 26.250 382.022 Td /F1 9.8 Tf [(DYSF2/IM DYSF2\) or a complete absence of dysferlin protein \(DYSF3/IM DYSF3 and DYSF4/IM DYSF4\))] TJ ET 0.965 0.965 0.965 rg 26.250 145.069 555.000 227.072 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 372.141 m 581.250 372.141 l 581.250 371.391 l 26.250 371.391 l f 26.250 145.069 m 581.250 145.069 l 581.250 145.819 l 26.250 145.819 l f q 225.000 0 0 92.250 35.250 270.141 cm /I3 Do Q q 35.250 156.319 537.000 107.822 re W n 0.271 0.267 0.267 rg BT 35.250 254.617 Td /F4 9.8 Tf [(Fig. 1: Comparison of immortalized human myoblasts and myotubes to their parental counterparts.)] TJ ET BT 35.250 235.247 Td /F1 9.8 Tf [(The expression of myogenic differentiation markers was analysed by Western blot in dysferlin-deficient and wild-type )] TJ ET BT 35.250 221.511 Td /F1 9.8 Tf [(human myoblasts and myotubes in primary cells \(A\) and after immortalization \(B\). A similar increase in MHC \(200kDa\) and )] TJ ET BT 35.250 207.775 Td /F1 9.8 Tf [(caveolin3 \(23kDa\) was observed in primary \(A\) and immortalized \(IM\) \(B\) cell lines after differentiation into myotubes. )] TJ ET BT 35.250 194.038 Td /F1 9.8 Tf [(Mutations in dysferlin result in a reduced expression \(IM DYSF1 and DYSF2/IM DYSF2\) or in a complete absence of )] TJ ET BT 35.250 180.302 Td /F1 9.8 Tf [(dysferlin protein \(230kDa\) \(DYSF3/IM DYSF3 and DYSF4/IM DYSF4\) in both myoblasts and myotubes. a-tubulin \(50kDa\) )] TJ ET BT 35.250 166.566 Td /F1 9.8 Tf [(has been used as a loading control.)] TJ ET Q BT 26.250 128.045 Td /F1 9.8 Tf [(In primary and immortalized wild-type myotubes and in IM DYSF1 and DYSF2/IM DYSF2 myotubes dysferlin was only )] TJ ET BT 26.250 116.140 Td /F1 9.8 Tf [(expressed as a single 230 kDa protein band as revealed by the HAMLET antibody directed against the C-terminal )] TJ ET BT 26.250 104.235 Td /F1 9.8 Tf [(juxtamembrane region of dysferlin and by a polyclonal antibody to the N-terminal part of dysferlin)] TJ ET 0.267 0.267 0.267 rg BT 442.965 104.235 Td /F1 9.8 Tf [( [10] )] TJ ET 0.271 0.267 0.267 rg BT 464.649 104.235 Td /F1 9.8 Tf [(\(data not shown\). This )] TJ ET BT 26.250 92.331 Td /F1 9.8 Tf [(suggests that dysferlin protein with disease causing-point mutations is expressed as the full-length protein.)] TJ ET BT 26.250 72.926 Td /F1 9.8 Tf [(Altogether dysferlin-deficient immortalized human myoblasts showed a differentiation pattern comparable to their untransformed )] TJ ET BT 26.250 61.021 Td /F1 9.8 Tf [(counterparts. Furthermore, the differentiation of myoblasts with disease-causing mutations in dysferlin into myotubes is similar )] TJ ET BT 26.250 49.116 Td /F1 9.8 Tf [(to wild-type myoblasts with respect to the expression of myogenic differentiation markers such as desmin, MHC and caveolin3.)] TJ ET Q q 225.000 0 0 92.250 35.250 270.141 cm /I3 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(3)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Muscular Dystrophy)] TJ ET Q endstream endobj 133 0 obj << /Type /Annot /Subtype /Link /A 134 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 270.1410 260.2500 362.3910 ] >> endobj 134 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/md/files/2012/02/fig1neu.tif) >> endobj 135 0 obj << /Type /XObject /Subtype /Image /Width 300 /Height 123 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 13230>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   {," }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?ǖc_Iʋ{ޙeY=6Mٖb6~P o[>#㫛&: +X.8G1e;Նf#}']|i&U쬢DP\[I{+᷅|C,ZeE,F\2B@E$=7ÚƱjQCw])a 1'U70#u4WOjm'}C[ibhT67B!4ߌ Դ{]2Ke`)Q΄` v}SJsRm>ĺe>ʐnO-$c??*G]_jZJ8Wš]֥ouu<XkV 8C<9[v$xSL8I[(oygQPGEqw_<i-u6/ UsF`N@ $\|!b(X蟷EOcdx=@:Z* mN ;.#Ya# + þ,|sּZSxF6Ś1yhU'ٯ@A~(5|S6tu(Li;.ڻm7#)+>*|'0xFҼZ%S d{bqWm7^;Pmm'-$/3E'AӠ蘒}kĭ ~&t=RKkB diAn͵#PSErWſ  ,4hD$N"[Õ<"<-ݼŵͭ~mR(7 t_hyZ;yEVp7$+ xþ3Rt=jSa svӫh(YAzQ@W=^@UX8F2#>Y#ր:+~,Wu:ŵA3J*>c~HMcTut۫"r |1f dutW`^m{:xL,x']ƍYLY ~SGQ[ZO=~Ll5 tY%$xՆU)$ z( (hh4[- OCNքFM`fINAʖAGk]2EKk-oΡ|./,a]H❽dHt(48fCk@l/$?" 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FGCjӡ.s"ѨHF3xPnywx l:m*ϊ4}-*&y_idTtoQGm啟SBQ7 pX@xtuE#\G::C)MxF,> HO?cD[<큧QxAJռr#e9r??Ꟶ~czZxVZg݊%F@8#u%K3h~ tg=6KS#;aGc]3C93]\4Ew4k-|Qy[{[Iq%ĦcJ7ra}=5_^عI7\# `"M˼b3' k  €Q}}icl]g,@IaFy\?~yz׷:Xink0PG\`>jk=m׶ża: NV"Nw|9>jP@U0S-ugxokvybY1Bʰ9ЎEh7H^P[Oj]\x^-mFIR>`HP+>?A1|M∡/ jJMp>zr5:L*%xCUP 7ܗz=*Ec Y"R2 ?{[-^_Fbn%$TD ?(yQ$6d|:AEP?mR"PDSڲ.<aq@e5ka3] X1<7:}ZnN2c *ڶZ$lҼ_^y-B}#a$i>GaLԊXc5qeh9yydJ x/XCRwkJC,):m%~:b}5ѵE}_٦͔e861[2xDk+nT?#yދoG Cbgs5 :/,МɓzZ+k#2Jų?'5ZbdzV2t4k^k:uZ[ k{9r3'A븼9ZEmf;䍗P V &r N3) <>8 GT5nel!HFI79QG>>LB@LFO ss]Z1'iݩ?C@ۍKWĺł˃{g_tlG^idӭ`d+ecܚrI^ \R*FA2񷆵O5{IshVv3O=~bܞ08iiZ5X/MܯhS#ث\\NYIk#3)QIg5M[*_]]-t֐1'vn|qZ^tf'Ԛq=#9O(|3/cDV[kYVFFA /kqgޡ&p s$io =c2bc>SnPhƮhVЮE: {gJ$d`4KBdܟm?hy.KGL&ȧَwW'9G#ij?ܬJ])Zl4-S՞H%V])3VS%ePj-=t1#zAKi$<|EGWI' HЭ 0Zz]gx=kIg;?kG5Ώ`h"²aWJ,ȷL ϯҀފ-ZYT;a†P1ru%3K9HdhcC43GLgHz`? endstream endobj 136 0 obj << /Type /Annot /Subtype /Link /A 137 0 R /Border [0 0 0] /H /I /Rect [ 442.9650 103.3336 464.6490 113.2542 ] >> endobj 137 0 obj << /Type /Action >> endobj 138 0 obj << /Type /Annot /Subtype /Link /A 139 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 270.1410 260.2500 362.3910 ] >> endobj 139 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/md/files/2012/02/fig1neu.tif) >> endobj 140 0 obj << /Type /Annot /Subtype /Link /A 141 0 R /Border [0 0 0] /H /I /Rect [ 442.9650 103.3336 464.6490 113.2542 ] >> endobj 141 0 obj << /Type /Action >> endobj 142 0 obj << /Type /Annot /Subtype /Link /A 143 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 270.1410 260.2500 362.3910 ] >> endobj 143 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/md/files/2012/02/fig1neu.tif) >> endobj 144 0 obj << /Type /Annot /Subtype /Link /A 145 0 R /Border [0 0 0] /H /I /Rect [ 442.9650 103.3336 464.6490 113.2542 ] >> endobj 145 0 obj << /Type /Action >> endobj 146 0 obj << /Type /Page /Parent 3 0 R /Annots [ 148 0 R 151 0 R 154 0 R 156 0 R 158 0 R 160 0 R ] /Contents 147 0 R >> endobj 147 0 obj << /Length 12142 >> stream 0.271 0.267 0.267 rg q 15.000 -13.481 577.500 790.481 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Differentiation pattern of dysferlin-deficient and wild-type immortalized human myoblasts)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Myogenic differentiation of dysferlin-deficient and wild-type immortalized human myoblasts into myotubes was followed by )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(immunocytochemistry to reveal the expression of early and late myogenic differentiation markers including the myogenic )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(regulatory transcription factors MyoD and myogenin, muscle structural proteins MHC, a-actinin and desmin and differentiation-)] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(regulated sarcolemmal proteins dysferlin and caveolin3. Representative examples are shown in Figure 2. After 7 days of )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(differentiation multinucleated myotubes were formed from dysferlin-deficient and from wild-type immortalized human myoblasts )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(as demonstrated by the expression of MHC, a-actinin and caveolin3 \(Fig.2\). Dysferlin was present in wild-type myotubes, )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(showed a reduced expression in IM DYSF1 and IM DYSF2 and was completely absent in IM DYSF3 and IM DYSF4 \(Fig.2\).)] TJ ET 0.965 0.965 0.965 rg 26.250 345.007 555.000 321.755 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 666.762 m 581.250 666.762 l 581.250 666.012 l 26.250 666.012 l f 26.250 345.007 m 581.250 345.007 l 581.250 345.757 l 26.250 345.757 l f q 225.000 0 0 202.500 35.250 454.512 cm /I4 Do Q q 35.250 356.257 537.000 92.255 re W n 0.271 0.267 0.267 rg BT 35.250 438.988 Td /F4 9.8 Tf [(Fig. 2: Differentiation state of myotubes derived from human immortalized dysferlin-deficient and wild-type )] TJ ET BT 35.250 427.083 Td /F4 9.8 Tf [(myoblasts.)] TJ ET BT 35.250 407.713 Td /F1 9.8 Tf [(Immunofluorescence stainings show the formation of multinucleated myotubes expressing the myogenic differentiation )] TJ ET BT 35.250 393.977 Td /F1 9.8 Tf [(markers desmin, a-actinin, MHC and caveolin3 in both wild-type and dysferlin-deficient myotubes. Disease-causing )] TJ ET BT 35.250 380.241 Td /F1 9.8 Tf [(mutations in dysferlin result in a reduced expression \(IMDYSF1 and IM DYSF2\) or in a complete absence of dysferlin \(IM )] TJ ET BT 35.250 366.505 Td /F1 9.8 Tf [(DYSF3 and IM DYSF4\). \(bar, 50m\))] TJ ET Q BT 26.250 327.984 Td /F1 9.8 Tf [(The subcellular localization of muscle differentiation-associated proteins was analyzed by high-resolution laser scanning )] TJ ET BT 26.250 316.079 Td /F1 9.8 Tf [(confocal microscopy. After 7 days of differentiation a striated staining pattern for MHC and a-actinin, labelling Z-lines, was )] TJ ET BT 26.250 304.174 Td /F1 9.8 Tf [(observed in myotubes derived from wild-type and dysferlin-deficient immortalized myoblasts \(Fig.3\). This is indicative of the )] TJ ET BT 26.250 292.269 Td /F1 9.8 Tf [(correct formation of sarcomeres in these myotubes independent of the presence or absence of dysferlin. Accordingly, )] TJ ET BT 26.250 280.365 Td /F1 9.8 Tf [(spontaneous contractions of myotubes were occasionally observed.)] TJ ET BT 26.250 260.960 Td /F1 9.8 Tf [(In wild-type myotubes dysferlin was distributed in the plasma membrane and the reticulate structures throughout the cell. )] TJ ET BT 26.250 249.055 Td /F1 9.8 Tf [(Myotubes with disease-causing mutations in dysferlin showed an intracellular aggregation \(IM DYSF1 and IM DYSF2\) or a )] TJ ET BT 26.250 237.150 Td /F1 9.8 Tf [(complete absence of dysferlin protein \(IM DYSF3 and IM DYSF4\) \(Fig.3\))] TJ ET 0.965 0.965 0.965 rg 26.250 -13.481 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 227.269 m 581.250 227.269 l 581.250 226.519 l 26.250 226.519 l f q 184.500 0 0 225.000 35.250 -7.481 cm /I5 Do Q q 35.250 -13.481 537.000 0.000 re W n Q Q q 15.000 -13.481 577.500 790.481 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Differentiation pattern of dysferlin-deficient and wild-type immortalized human myoblasts)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Myogenic differentiation of dysferlin-deficient and wild-type immortalized human myoblasts into myotubes was followed by )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(immunocytochemistry to reveal the expression of early and late myogenic differentiation markers including the myogenic )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(regulatory transcription factors MyoD and myogenin, muscle structural proteins MHC, a-actinin and desmin and differentiation-)] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(regulated sarcolemmal proteins dysferlin and caveolin3. Representative examples are shown in Figure 2. After 7 days of )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(differentiation multinucleated myotubes were formed from dysferlin-deficient and from wild-type immortalized human myoblasts )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(as demonstrated by the expression of MHC, a-actinin and caveolin3 \(Fig.2\). Dysferlin was present in wild-type myotubes, )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(showed a reduced expression in IM DYSF1 and IM DYSF2 and was completely absent in IM DYSF3 and IM DYSF4 \(Fig.2\).)] TJ ET 0.965 0.965 0.965 rg 26.250 345.007 555.000 321.755 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 666.762 m 581.250 666.762 l 581.250 666.012 l 26.250 666.012 l f 26.250 345.007 m 581.250 345.007 l 581.250 345.757 l 26.250 345.757 l f q 225.000 0 0 202.500 35.250 454.512 cm /I4 Do Q q 35.250 356.257 537.000 92.255 re W n 0.271 0.267 0.267 rg BT 35.250 438.988 Td /F4 9.8 Tf [(Fig. 2: Differentiation state of myotubes derived from human immortalized dysferlin-deficient and wild-type )] TJ ET BT 35.250 427.083 Td /F4 9.8 Tf [(myoblasts.)] TJ ET BT 35.250 407.713 Td /F1 9.8 Tf [(Immunofluorescence stainings show the formation of multinucleated myotubes expressing the myogenic differentiation )] TJ ET BT 35.250 393.977 Td /F1 9.8 Tf [(markers desmin, a-actinin, MHC and caveolin3 in both wild-type and dysferlin-deficient myotubes. Disease-causing )] TJ ET BT 35.250 380.241 Td /F1 9.8 Tf [(mutations in dysferlin result in a reduced expression \(IMDYSF1 and IM DYSF2\) or in a complete absence of dysferlin \(IM )] TJ ET BT 35.250 366.505 Td /F1 9.8 Tf [(DYSF3 and IM DYSF4\). \(bar, 50m\))] TJ ET Q BT 26.250 327.984 Td /F1 9.8 Tf [(The subcellular localization of muscle differentiation-associated proteins was analyzed by high-resolution laser scanning )] TJ ET BT 26.250 316.079 Td /F1 9.8 Tf [(confocal microscopy. After 7 days of differentiation a striated staining pattern for MHC and a-actinin, labelling Z-lines, was )] TJ ET BT 26.250 304.174 Td /F1 9.8 Tf [(observed in myotubes derived from wild-type and dysferlin-deficient immortalized myoblasts \(Fig.3\). This is indicative of the )] TJ ET BT 26.250 292.269 Td /F1 9.8 Tf [(correct formation of sarcomeres in these myotubes independent of the presence or absence of dysferlin. Accordingly, )] TJ ET BT 26.250 280.365 Td /F1 9.8 Tf [(spontaneous contractions of myotubes were occasionally observed.)] TJ ET BT 26.250 260.960 Td /F1 9.8 Tf [(In wild-type myotubes dysferlin was distributed in the plasma membrane and the reticulate structures throughout the cell. )] TJ ET BT 26.250 249.055 Td /F1 9.8 Tf [(Myotubes with disease-causing mutations in dysferlin showed an intracellular aggregation \(IM DYSF1 and IM DYSF2\) or a )] TJ ET BT 26.250 237.150 Td /F1 9.8 Tf [(complete absence of dysferlin protein \(IM DYSF3 and IM DYSF4\) \(Fig.3\))] TJ ET 0.965 0.965 0.965 rg 26.250 -13.481 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 227.269 m 581.250 227.269 l 581.250 226.519 l 26.250 226.519 l f q 184.500 0 0 225.000 35.250 -7.481 cm /I5 Do Q q 35.250 -13.481 537.000 0.000 re W n Q Q q 15.000 -13.481 577.500 790.481 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F4 9.8 Tf [(Differentiation pattern of dysferlin-deficient and wild-type immortalized human myoblasts)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Myogenic differentiation of dysferlin-deficient and wild-type immortalized human myoblasts into myotubes was followed by )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(immunocytochemistry to reveal the expression of early and late myogenic differentiation markers including the myogenic )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(regulatory transcription factors MyoD and myogenin, muscle structural proteins MHC, a-actinin and desmin and differentiation-)] TJ ET BT 26.250 712.357 Td /F1 9.8 Tf [(regulated sarcolemmal proteins dysferlin and caveolin3. Representative examples are shown in Figure 2. After 7 days of )] TJ ET BT 26.250 700.452 Td /F1 9.8 Tf [(differentiation multinucleated myotubes were formed from dysferlin-deficient and from wild-type immortalized human myoblasts )] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(as demonstrated by the expression of MHC, a-actinin and caveolin3 \(Fig.2\). Dysferlin was present in wild-type myotubes, )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(showed a reduced expression in IM DYSF1 and IM DYSF2 and was completely absent in IM DYSF3 and IM DYSF4 \(Fig.2\).)] TJ ET 0.965 0.965 0.965 rg 26.250 345.007 555.000 321.755 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 666.762 m 581.250 666.762 l 581.250 666.012 l 26.250 666.012 l f 26.250 345.007 m 581.250 345.007 l 581.250 345.757 l 26.250 345.757 l f q 225.000 0 0 202.500 35.250 454.512 cm /I4 Do Q q 35.250 356.257 537.000 92.255 re W n 0.271 0.267 0.267 rg BT 35.250 438.988 Td /F4 9.8 Tf [(Fig. 2: Differentiation state of myotubes derived from human immortalized dysferlin-deficient and wild-type )] TJ ET BT 35.250 427.083 Td /F4 9.8 Tf [(myoblasts.)] TJ ET BT 35.250 407.713 Td /F1 9.8 Tf [(Immunofluorescence stainings show the formation of multinucleated myotubes expressing the myogenic differentiation )] TJ ET BT 35.250 393.977 Td /F1 9.8 Tf [(markers desmin, a-actinin, MHC and caveolin3 in both wild-type and dysferlin-deficient myotubes. Disease-causing )] TJ ET BT 35.250 380.241 Td /F1 9.8 Tf [(mutations in dysferlin result in a reduced expression \(IMDYSF1 and IM DYSF2\) or in a complete absence of dysferlin \(IM )] TJ ET BT 35.250 366.505 Td /F1 9.8 Tf [(DYSF3 and IM DYSF4\). \(bar, 50m\))] TJ ET Q BT 26.250 327.984 Td /F1 9.8 Tf [(The subcellular localization of muscle differentiation-associated proteins was analyzed by high-resolution laser scanning )] TJ ET BT 26.250 316.079 Td /F1 9.8 Tf [(confocal microscopy. After 7 days of differentiation a striated staining pattern for MHC and a-actinin, labelling Z-lines, was )] TJ ET BT 26.250 304.174 Td /F1 9.8 Tf [(observed in myotubes derived from wild-type and dysferlin-deficient immortalized myoblasts \(Fig.3\). This is indicative of the )] TJ ET BT 26.250 292.269 Td /F1 9.8 Tf [(correct formation of sarcomeres in these myotubes independent of the presence or absence of dysferlin. Accordingly, )] TJ ET BT 26.250 280.365 Td /F1 9.8 Tf [(spontaneous contractions of myotubes were occasionally observed.)] TJ ET BT 26.250 260.960 Td /F1 9.8 Tf [(In wild-type myotubes dysferlin was distributed in the plasma membrane and the reticulate structures throughout the cell. )] TJ ET BT 26.250 249.055 Td /F1 9.8 Tf [(Myotubes with disease-causing mutations in dysferlin showed an intracellular aggregation \(IM DYSF1 and IM DYSF2\) or a )] TJ ET BT 26.250 237.150 Td /F1 9.8 Tf [(complete absence of dysferlin protein \(IM DYSF3 and IM DYSF4\) \(Fig.3\))] TJ ET 0.965 0.965 0.965 rg 26.250 -13.481 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 227.269 m 581.250 227.269 l 581.250 226.519 l 26.250 226.519 l f q 184.500 0 0 225.000 35.250 -7.481 cm /I5 Do Q q 35.250 -13.481 537.000 0.000 re W n Q Q q 225.000 0 0 202.500 35.250 454.512 cm /I4 Do Q q 184.500 0 0 225.000 35.250 -7.481 cm /I5 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(4)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Muscular Dystrophy)] TJ ET Q endstream endobj 148 0 obj << /Type /Annot /Subtype /Link /A 149 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 454.5120 260.2500 657.0120 ] >> endobj 149 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/md/files/2012/02/fig2neu.tif) >> endobj 150 0 obj << /Type /XObject /Subtype /Image /Width 300 /Height 270 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 33317>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   ," }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Q/I#ÉY5-HiO<lc7a6ϡ>'zŚ\jMf8Č'X]CqJҿpaCk,n k03tU,^Z|.4d8Y:[X1+:ƟxFtѣ'mx>i-UH[YUfuÐ5խ-9" 0s62>}G ׬.,W"B*xK^N %ڻYAHS9`@=fXoγL#崯Ͷ[E;W_+OPvImZ8eF>i#s1\G>5&V v9${8jrŧ*:)\[pw>]G|כx⇋_|1_q,ZE#Gm_2ԙ |S_ iof:N i}(d r?ZM{ ۛ>5iM:+eKH'kϒ? 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Dysferlin was distributed to the plasma membrane in wild-type )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(immortalized myotubes \(IM WT2\). Disease-causing mutations in dysferlin result in an intracellular aggregation \(IM DYSF1 )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(and IM DYSF2\) or a complete absence of dysferlin protein \(IM DYSF3 and IM DYSF4\). \(bar 5m, left panels; bar 20m, )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(right panels\))] TJ ET Q BT 26.250 642.735 Td /F4 9.8 Tf [(Dysferlin-deficiency and membrane repair)] TJ ET BT 26.250 623.331 Td /F1 9.8 Tf [(Since dysferlin is involved in plasma membrane resealing after injury, laser wounding assays were performed. Myotubes )] TJ ET BT 26.250 611.426 Td /F1 9.8 Tf [(derived from dysferlin-deficient and wild-type immortalized human myoblasts were compared with respect to membrane repair. )] TJ ET BT 26.250 599.521 Td /F1 9.8 Tf [(Membrane wounding was conducted employing a laser confocal microscope and the fluorescent membrane dye FM1-43 was )] TJ ET BT 26.250 587.616 Td /F1 9.8 Tf [(used as a readout of membrane repair, as described by Cai et al. )] TJ ET 0.267 0.267 0.267 rg BT 309.673 587.616 Td /F1 9.8 Tf [([14])] TJ ET 0.271 0.267 0.267 rg BT 325.936 587.616 Td /F1 9.8 Tf [( . In myotubes derived from dysferlin-deficient )] TJ ET BT 26.250 575.712 Td /F1 9.8 Tf [(immortalized myoblasts, we observed an opening of the targeted membrane frontier and a staining of intracellular membrane )] TJ ET BT 26.250 563.807 Td /F1 9.8 Tf [(compartments by the influx of the fluorescent dye FM1-43 \(Fig.4B and 4C\). Membrane integrity was not restored during a 280 )] TJ ET BT 26.250 551.902 Td /F1 9.8 Tf [(sec observation period. In myotubes from wild-type immortalized human myoblasts, we observed resealing of the targeted )] TJ ET BT 26.250 539.997 Td /F1 9.8 Tf [(membrane frontier after laser wounding and no influx of FM1-43 during the remaining 280 sec \(Fig.4A\). These observations )] TJ ET BT 26.250 528.093 Td /F1 9.8 Tf [(were reinforced by the quantification of the increase in fluorescence intensity in dysferlin-deficient compared to wild-type )] TJ ET BT 26.250 516.188 Td /F1 9.8 Tf [(myotubes \(Fig.4D\).)] TJ ET 0.965 0.965 0.965 rg 26.250 160.221 555.000 346.086 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 506.307 m 581.250 506.307 l 581.250 505.557 l 26.250 505.557 l f 26.250 160.221 m 581.250 160.221 l 581.250 160.971 l 26.250 160.971 l f q 225.000 0 0 225.000 35.250 271.557 cm /I6 Do Q q 35.250 171.471 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 256.033 Td /F4 9.8 Tf [(Fig. 4: Laser mediated membrane wounding as an assay for dysferlin protein function.)] TJ ET BT 35.250 236.663 Td /F1 9.8 Tf [(Myotubes derived from immortalized human myoblasts were wounded by laser irradiation and FM1-43 influx was visualized )] TJ ET BT 35.250 222.927 Td /F1 9.8 Tf [(every 20 sec. Dysferlin-deficient myotubes \(B and C; IM DYSF1 and IM DYSF2\) show an influx of FM1-43 after membrane )] TJ ET BT 35.250 209.191 Td /F1 9.8 Tf [(wounding that was not observed in wild-type myotubes \(A; IM WT3\). Summary data for FM1-43 dye entry into myotubes )] TJ ET BT 35.250 195.454 Td /F1 9.8 Tf [(following laser mediated membrane wounding are shown in \(D\). Data represent means SE \(n=7, IM WT3; n= 8, IM )] TJ ET BT 35.250 181.718 Td /F1 9.8 Tf [(DYSF1; n=11, IM DYSF2\). \(bar, 5m\))] TJ ET Q BT 26.250 125.999 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 106.045 Td /F1 9.8 Tf [(Primary human myoblasts are an indispensible tool to study cellular processes in muscle disease. However, the material is very )] TJ ET BT 26.250 94.140 Td /F1 9.8 Tf [(precious and available in very small quantities because of the limited size of biopsy procedures that can be performed and the )] TJ ET BT 26.250 82.236 Td /F1 9.8 Tf [(reduced proliferative potential of human myoblasts which will be further reduced in muscular dystrophies due to the cycles of )] TJ ET BT 26.250 70.331 Td /F1 9.8 Tf [(degeneration and regeneration. Furthermore, primary cultures from dystrophic muscle are generally highly intermingled with )] TJ ET BT 26.250 58.426 Td /F1 9.8 Tf [(fibroblasts that cannot always be sorted completely. In the past, we observed that cultures derived from different LGMD2B )] TJ ET BT 26.250 46.521 Td /F1 9.8 Tf [(patients consisted mainly of fibroblasts and adipocytes and we were therefore unable to generate pure primary myoblast lines )] TJ ET Q q 15.000 32.236 577.500 744.764 re W n 0.965 0.965 0.965 rg 26.250 659.759 555.000 117.241 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 659.759 m 581.250 659.759 l 581.250 660.509 l 26.250 660.509 l f q 35.250 671.009 537.000 105.991 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Subcellular localization of muscle differentiation-specific proteins in myotubes derived from human )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(immortalized dysferlin-deficient and wild-type clones of myoblasts.)] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(Immunofluorescence staining with either anti-MHC or with anti-?-actinin antibody revealed correct organization of the )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(sarcomere and Z-lines, respectively, in all cell lines. Dysferlin was distributed to the plasma membrane in wild-type )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(immortalized myotubes \(IM WT2\). Disease-causing mutations in dysferlin result in an intracellular aggregation \(IM DYSF1 )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(and IM DYSF2\) or a complete absence of dysferlin protein \(IM DYSF3 and IM DYSF4\). \(bar 5m, left panels; bar 20m, )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(right panels\))] TJ ET Q BT 26.250 642.735 Td /F4 9.8 Tf [(Dysferlin-deficiency and membrane repair)] TJ ET BT 26.250 623.331 Td /F1 9.8 Tf [(Since dysferlin is involved in plasma membrane resealing after injury, laser wounding assays were performed. Myotubes )] TJ ET BT 26.250 611.426 Td /F1 9.8 Tf [(derived from dysferlin-deficient and wild-type immortalized human myoblasts were compared with respect to membrane repair. )] TJ ET BT 26.250 599.521 Td /F1 9.8 Tf [(Membrane wounding was conducted employing a laser confocal microscope and the fluorescent membrane dye FM1-43 was )] TJ ET BT 26.250 587.616 Td /F1 9.8 Tf [(used as a readout of membrane repair, as described by Cai et al. )] TJ ET 0.267 0.267 0.267 rg BT 309.673 587.616 Td /F1 9.8 Tf [([14])] TJ ET 0.271 0.267 0.267 rg BT 325.936 587.616 Td /F1 9.8 Tf [( . In myotubes derived from dysferlin-deficient )] TJ ET BT 26.250 575.712 Td /F1 9.8 Tf [(immortalized myoblasts, we observed an opening of the targeted membrane frontier and a staining of intracellular membrane )] TJ ET BT 26.250 563.807 Td /F1 9.8 Tf [(compartments by the influx of the fluorescent dye FM1-43 \(Fig.4B and 4C\). Membrane integrity was not restored during a 280 )] TJ ET BT 26.250 551.902 Td /F1 9.8 Tf [(sec observation period. In myotubes from wild-type immortalized human myoblasts, we observed resealing of the targeted )] TJ ET BT 26.250 539.997 Td /F1 9.8 Tf [(membrane frontier after laser wounding and no influx of FM1-43 during the remaining 280 sec \(Fig.4A\). These observations )] TJ ET BT 26.250 528.093 Td /F1 9.8 Tf [(were reinforced by the quantification of the increase in fluorescence intensity in dysferlin-deficient compared to wild-type )] TJ ET BT 26.250 516.188 Td /F1 9.8 Tf [(myotubes \(Fig.4D\).)] TJ ET 0.965 0.965 0.965 rg 26.250 160.221 555.000 346.086 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 506.307 m 581.250 506.307 l 581.250 505.557 l 26.250 505.557 l f 26.250 160.221 m 581.250 160.221 l 581.250 160.971 l 26.250 160.971 l f q 225.000 0 0 225.000 35.250 271.557 cm /I6 Do Q q 35.250 171.471 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 256.033 Td /F4 9.8 Tf [(Fig. 4: Laser mediated membrane wounding as an assay for dysferlin protein function.)] TJ ET BT 35.250 236.663 Td /F1 9.8 Tf [(Myotubes derived from immortalized human myoblasts were wounded by laser irradiation and FM1-43 influx was visualized )] TJ ET BT 35.250 222.927 Td /F1 9.8 Tf [(every 20 sec. Dysferlin-deficient myotubes \(B and C; IM DYSF1 and IM DYSF2\) show an influx of FM1-43 after membrane )] TJ ET BT 35.250 209.191 Td /F1 9.8 Tf [(wounding that was not observed in wild-type myotubes \(A; IM WT3\). Summary data for FM1-43 dye entry into myotubes )] TJ ET BT 35.250 195.454 Td /F1 9.8 Tf [(following laser mediated membrane wounding are shown in \(D\). Data represent means SE \(n=7, IM WT3; n= 8, IM )] TJ ET BT 35.250 181.718 Td /F1 9.8 Tf [(DYSF1; n=11, IM DYSF2\). \(bar, 5m\))] TJ ET Q BT 26.250 125.999 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 106.045 Td /F1 9.8 Tf [(Primary human myoblasts are an indispensible tool to study cellular processes in muscle disease. However, the material is very )] TJ ET BT 26.250 94.140 Td /F1 9.8 Tf [(precious and available in very small quantities because of the limited size of biopsy procedures that can be performed and the )] TJ ET BT 26.250 82.236 Td /F1 9.8 Tf [(reduced proliferative potential of human myoblasts which will be further reduced in muscular dystrophies due to the cycles of )] TJ ET BT 26.250 70.331 Td /F1 9.8 Tf [(degeneration and regeneration. Furthermore, primary cultures from dystrophic muscle are generally highly intermingled with )] TJ ET BT 26.250 58.426 Td /F1 9.8 Tf [(fibroblasts that cannot always be sorted completely. In the past, we observed that cultures derived from different LGMD2B )] TJ ET BT 26.250 46.521 Td /F1 9.8 Tf [(patients consisted mainly of fibroblasts and adipocytes and we were therefore unable to generate pure primary myoblast lines )] TJ ET Q q 15.000 32.236 577.500 744.764 re W n 0.965 0.965 0.965 rg 26.250 659.759 555.000 117.241 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 659.759 m 581.250 659.759 l 581.250 660.509 l 26.250 660.509 l f q 35.250 671.009 537.000 105.991 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Subcellular localization of muscle differentiation-specific proteins in myotubes derived from human )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(immortalized dysferlin-deficient and wild-type clones of myoblasts.)] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(Immunofluorescence staining with either anti-MHC or with anti-?-actinin antibody revealed correct organization of the )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(sarcomere and Z-lines, respectively, in all cell lines. Dysferlin was distributed to the plasma membrane in wild-type )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(immortalized myotubes \(IM WT2\). Disease-causing mutations in dysferlin result in an intracellular aggregation \(IM DYSF1 )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(and IM DYSF2\) or a complete absence of dysferlin protein \(IM DYSF3 and IM DYSF4\). \(bar 5m, left panels; bar 20m, )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(right panels\))] TJ ET Q BT 26.250 642.735 Td /F4 9.8 Tf [(Dysferlin-deficiency and membrane repair)] TJ ET BT 26.250 623.331 Td /F1 9.8 Tf [(Since dysferlin is involved in plasma membrane resealing after injury, laser wounding assays were performed. Myotubes )] TJ ET BT 26.250 611.426 Td /F1 9.8 Tf [(derived from dysferlin-deficient and wild-type immortalized human myoblasts were compared with respect to membrane repair. )] TJ ET BT 26.250 599.521 Td /F1 9.8 Tf [(Membrane wounding was conducted employing a laser confocal microscope and the fluorescent membrane dye FM1-43 was )] TJ ET BT 26.250 587.616 Td /F1 9.8 Tf [(used as a readout of membrane repair, as described by Cai et al. )] TJ ET 0.267 0.267 0.267 rg BT 309.673 587.616 Td /F1 9.8 Tf [([14])] TJ ET 0.271 0.267 0.267 rg BT 325.936 587.616 Td /F1 9.8 Tf [( . In myotubes derived from dysferlin-deficient )] TJ ET BT 26.250 575.712 Td /F1 9.8 Tf [(immortalized myoblasts, we observed an opening of the targeted membrane frontier and a staining of intracellular membrane )] TJ ET BT 26.250 563.807 Td /F1 9.8 Tf [(compartments by the influx of the fluorescent dye FM1-43 \(Fig.4B and 4C\). Membrane integrity was not restored during a 280 )] TJ ET BT 26.250 551.902 Td /F1 9.8 Tf [(sec observation period. In myotubes from wild-type immortalized human myoblasts, we observed resealing of the targeted )] TJ ET BT 26.250 539.997 Td /F1 9.8 Tf [(membrane frontier after laser wounding and no influx of FM1-43 during the remaining 280 sec \(Fig.4A\). These observations )] TJ ET BT 26.250 528.093 Td /F1 9.8 Tf [(were reinforced by the quantification of the increase in fluorescence intensity in dysferlin-deficient compared to wild-type )] TJ ET BT 26.250 516.188 Td /F1 9.8 Tf [(myotubes \(Fig.4D\).)] TJ ET 0.965 0.965 0.965 rg 26.250 160.221 555.000 346.086 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 506.307 m 581.250 506.307 l 581.250 505.557 l 26.250 505.557 l f 26.250 160.221 m 581.250 160.221 l 581.250 160.971 l 26.250 160.971 l f q 225.000 0 0 225.000 35.250 271.557 cm /I6 Do Q q 35.250 171.471 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 256.033 Td /F4 9.8 Tf [(Fig. 4: Laser mediated membrane wounding as an assay for dysferlin protein function.)] TJ ET BT 35.250 236.663 Td /F1 9.8 Tf [(Myotubes derived from immortalized human myoblasts were wounded by laser irradiation and FM1-43 influx was visualized )] TJ ET BT 35.250 222.927 Td /F1 9.8 Tf [(every 20 sec. Dysferlin-deficient myotubes \(B and C; IM DYSF1 and IM DYSF2\) show an influx of FM1-43 after membrane )] TJ ET BT 35.250 209.191 Td /F1 9.8 Tf [(wounding that was not observed in wild-type myotubes \(A; IM WT3\). Summary data for FM1-43 dye entry into myotubes )] TJ ET BT 35.250 195.454 Td /F1 9.8 Tf [(following laser mediated membrane wounding are shown in \(D\). Data represent means SE \(n=7, IM WT3; n= 8, IM )] TJ ET BT 35.250 181.718 Td /F1 9.8 Tf [(DYSF1; n=11, IM DYSF2\). \(bar, 5m\))] TJ ET Q BT 26.250 125.999 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 106.045 Td /F1 9.8 Tf [(Primary human myoblasts are an indispensible tool to study cellular processes in muscle disease. However, the material is very )] TJ ET BT 26.250 94.140 Td /F1 9.8 Tf [(precious and available in very small quantities because of the limited size of biopsy procedures that can be performed and the )] TJ ET BT 26.250 82.236 Td /F1 9.8 Tf [(reduced proliferative potential of human myoblasts which will be further reduced in muscular dystrophies due to the cycles of )] TJ ET BT 26.250 70.331 Td /F1 9.8 Tf [(degeneration and regeneration. Furthermore, primary cultures from dystrophic muscle are generally highly intermingled with )] TJ ET BT 26.250 58.426 Td /F1 9.8 Tf [(fibroblasts that cannot always be sorted completely. 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However, in the present study it was possible to get a 100% myogenic )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(population of IM DYSF1 after immortalization with hTERT and CDK-4 and subsequent cloning highlighting the great potential of )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(the immortalization procedure. We thoroughly characterized four immortalized LGMD2B patient cell lines harbouring different )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(dysferlin mutations not only for myogenic differentiation but also functionally and found no significant differences to their )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(parental cell lines \(Fig.1 and data not shown\). Therefore, the immortalization of primary human myoblasts represents a major )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(advantage and may overcome the limitations mentioned above. In addition to LGMD2B, immortalized human myoblast lines )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(have been successfully established from patients with other muscle diseases including facioscapulohumeral MD, Duchenne )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(MD, congenital MD and oculo-pharyngeal MD )] TJ ET 0.267 0.267 0.267 rg BT 226.193 684.143 Td /F1 9.8 Tf [([13] [15])] TJ ET 0.271 0.267 0.267 rg BT 261.430 684.143 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(Human cell lines with unlimited proliferative capacity are useful tools in cell biology and for translational research. Up to now, )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(only murine myoblast lines are available that are deficient for dysferlin. 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Dysferlin-deficient myoblasts have been described to fully differentiate without any impairment as compared to wild-)] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(type myoblasts with respect to myoblast fusion and activation of myogenic pathways. This strongly suggests that dysferlin does )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(not play a role in early stages of myotube formation and subsequent maturation)] TJ ET 0.267 0.267 0.267 rg BT 368.212 573.905 Td /F1 9.8 Tf [( [7] [18] [19] [20] [21])] TJ ET 0.271 0.267 0.267 rg BT 457.658 573.905 Td /F1 9.8 Tf [( . Instead, myoferlin, )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(another member of the ferlin protein family, has been described to be essential for myoblast-myoblast and myoblast-myotube )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(fusion )] TJ ET 0.267 0.267 0.267 rg BT 54.974 550.095 Td /F1 9.8 Tf [([18] [22])] TJ ET 0.271 0.267 0.267 rg BT 90.210 550.095 Td /F1 9.8 Tf [( . Myoferlin is expressed early during myoblast differentiation whereas dysferlin is expressed only after the )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(formation of multinucleated myotubes )] TJ ET 0.267 0.267 0.267 rg BT 190.986 538.191 Td /F1 9.8 Tf [([18] [22])] TJ ET 0.271 0.267 0.267 rg BT 226.222 538.191 Td /F1 9.8 Tf [( . Contradictorily, a reduced or delayed differentiation of dysferlin-deficient )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(myoblasts into myotubes has been attributed to dysferlin-deficiency by other groups )] TJ ET 0.267 0.267 0.267 rg BT 389.330 526.286 Td /F1 9.8 Tf [([17] [23] [24])] TJ ET 0.271 0.267 0.267 rg BT 443.540 526.286 Td /F1 9.8 Tf [( . Reasons for these )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(discrepancies might be caused by the use of different cellular models, species, culture conditions, )] TJ ET BT 449.478 514.381 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 478.192 514.381 Td /F1 9.8 Tf [( cultivation time, )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(experimental design or developmental differences \(e.g. age of the donor patient\) that finally result in a different myogenic )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(potential and differentiation kinetics. For instance it has been shown that differentiation kinetics of immortalized myoblast lines )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(slow down with time in culture probably due to constant selection for proliferation )] TJ ET 0.267 0.267 0.267 rg BT 376.314 478.667 Td /F1 9.8 Tf [([15])] TJ ET 0.271 0.267 0.267 rg BT 392.577 478.667 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 459.262 Td /F1 9.8 Tf [(Being able to assess new therapeutical approaches is of great significance and requires to prove the proper function of the )] TJ ET BT 26.250 447.357 Td /F1 9.8 Tf [(restored protein. This can be achieved partially by analysis of the correct intracellular localisation and the size of the protein )] TJ ET BT 26.250 435.453 Td /F1 9.8 Tf [(using immunochemical approaches. In the case of dysferlin the assumption that dysferlin is indispensable in sarcolemmal repair )] TJ ET BT 26.250 423.548 Td /F1 9.8 Tf [(opens the possibility for a direct functional assay by laser-mediated membrane wounding in cultured myotubes and myofibers. )] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(We show here that myotubes derived from the immortalized dysferlin-deficient myoblast lines, e.g. IM DYSF1 and IM DYSF2, )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(can be employed as a read-out tool of dysferlin functionality by laser-mediated wounding of the sarcolemma. Our results are in )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(accordance with the earlier observed dysfunction of the membrane resealing process in the absence of dysferlin in myotubes )] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(and myofibers )] TJ ET 0.267 0.267 0.267 rg BT 90.191 375.929 Td /F1 9.8 Tf [([6] [8] [14])] TJ ET 0.271 0.267 0.267 rg BT 133.559 375.929 Td /F1 9.8 Tf [( . We conclude that the human immortalized dysferlin-deficient myoblast lines represent innovative )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(tools to assess dysferlin functionality after application of pharmacological and genetical approaches to restore dysferlin.)] TJ ET BT 26.250 344.619 Td /F1 9.8 Tf [(Although we did not analyze cellular metabolism and regulation of cell cycle progression we expect metabolic changes in )] TJ ET BT 26.250 332.715 Td /F1 9.8 Tf [(immortalized myoblasts due to their high proliferative potential. However, this seems to have no influence on myogenic )] TJ ET BT 26.250 320.810 Td /F1 9.8 Tf [(differentiation and dysferlin function in immortalized myoblasts and their corresponding myotubes as demonstrated in this report.)] TJ ET BT 26.250 301.405 Td /F1 9.8 Tf [(In summary, the immortalized myoblast cell lines display properties highly similar to their parental cell lines with respect to )] TJ ET BT 26.250 289.500 Td /F1 9.8 Tf [(myogenic differentiation, formation of multinucleated myotubes, development of a correct myofibrillar architecture and dysferlin )] TJ ET BT 26.250 277.596 Td /F1 9.8 Tf [(protein expression. Dysferlin reveals unaltered subcellular localization and function in membrane repair in control cell lines, )] TJ ET BT 26.250 265.691 Td /F1 9.8 Tf [(while it is perturbed in cell lines derived from LGMD-2B patients. In addition dysferlin-deficient myoblasts have been described )] TJ ET BT 26.250 253.786 Td /F1 9.8 Tf [(to fully differentiate suggesting that dysferlin does not play a role in early stages of myotube formation. Therefore, immortalized )] TJ ET BT 26.250 241.881 Td /F1 9.8 Tf [(human myoblast lines harbouring different mutations in dysferlin represent a very useful tool to further investigate dysferlin )] TJ ET BT 26.250 229.977 Td /F1 9.8 Tf [(function, to study the pathophysiological mechanisms involved in dysferlinopathy and more importantly to assess therapeutic )] TJ ET BT 26.250 218.072 Td /F1 9.8 Tf [(strategies to correct dysferlinopathies with a reliable readout.)] TJ ET BT 26.250 181.469 Td /F4 12.0 Tf [(Competing interests)] TJ ET BT 26.250 161.515 Td /F1 9.8 Tf [(The authors have declared that no competing interests exist.)] TJ ET BT 26.250 124.913 Td /F4 12.0 Tf [(Address for Correspondence)] TJ ET BT 26.250 104.958 Td /F1 9.8 Tf [(Simone Spuler, MD)] TJ ET BT 26.250 85.554 Td /F1 9.8 Tf [(ECRC, Charit Campus Buch, Lindenberger Weg 80,)] TJ ET BT 26.250 66.149 Td /F1 9.8 Tf [(13125 Berlin, Germany)] TJ ET Q q 15.000 36.863 577.500 740.137 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(from them, as examplified for DYSF1 in this report. However, in the present study it was possible to get a 100% myogenic )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(population of IM DYSF1 after immortalization with hTERT and CDK-4 and subsequent cloning highlighting the great potential of )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(the immortalization procedure. We thoroughly characterized four immortalized LGMD2B patient cell lines harbouring different )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(dysferlin mutations not only for myogenic differentiation but also functionally and found no significant differences to their )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(parental cell lines \(Fig.1 and data not shown\). Therefore, the immortalization of primary human myoblasts represents a major )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(advantage and may overcome the limitations mentioned above. In addition to LGMD2B, immortalized human myoblast lines )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(have been successfully established from patients with other muscle diseases including facioscapulohumeral MD, Duchenne )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(MD, congenital MD and oculo-pharyngeal MD )] TJ ET 0.267 0.267 0.267 rg BT 226.193 684.143 Td /F1 9.8 Tf [([13] [15])] TJ ET 0.271 0.267 0.267 rg BT 261.430 684.143 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(Human cell lines with unlimited proliferative capacity are useful tools in cell biology and for translational research. Up to now, )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(only murine myoblast lines are available that are deficient for dysferlin. GREG cells were derived from A/J mice lacking dysferlin )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(expression)] TJ ET 0.267 0.267 0.267 rg BT 73.391 640.929 Td /F1 9.8 Tf [( [16] )] TJ ET 0.271 0.267 0.267 rg BT 95.075 640.929 Td /F1 9.8 Tf [(and a C2C12 cell clone with a stable dysferlin knock-down by shRNA has been establihed that expresses about )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(10% of residual dysferlin )] TJ ET 0.267 0.267 0.267 rg BT 135.167 629.024 Td /F1 9.8 Tf [([17])] TJ ET 0.271 0.267 0.267 rg BT 151.430 629.024 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 609.619 Td /F1 9.8 Tf [(There are discrepancies in the literature about the potential of dysferlin-deficient myoblasts to fuse and to differentiate into )] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(myotubes. Dysferlin-deficient myoblasts have been described to fully differentiate without any impairment as compared to wild-)] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(type myoblasts with respect to myoblast fusion and activation of myogenic pathways. This strongly suggests that dysferlin does )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(not play a role in early stages of myotube formation and subsequent maturation)] TJ ET 0.267 0.267 0.267 rg BT 368.212 573.905 Td /F1 9.8 Tf [( [7] [18] [19] [20] [21])] TJ ET 0.271 0.267 0.267 rg BT 457.658 573.905 Td /F1 9.8 Tf [( . Instead, myoferlin, )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(another member of the ferlin protein family, has been described to be essential for myoblast-myoblast and myoblast-myotube )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(fusion )] TJ ET 0.267 0.267 0.267 rg BT 54.974 550.095 Td /F1 9.8 Tf [([18] [22])] TJ ET 0.271 0.267 0.267 rg BT 90.210 550.095 Td /F1 9.8 Tf [( . Myoferlin is expressed early during myoblast differentiation whereas dysferlin is expressed only after the )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(formation of multinucleated myotubes )] TJ ET 0.267 0.267 0.267 rg BT 190.986 538.191 Td /F1 9.8 Tf [([18] [22])] TJ ET 0.271 0.267 0.267 rg BT 226.222 538.191 Td /F1 9.8 Tf [( . Contradictorily, a reduced or delayed differentiation of dysferlin-deficient )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(myoblasts into myotubes has been attributed to dysferlin-deficiency by other groups )] TJ ET 0.267 0.267 0.267 rg BT 389.330 526.286 Td /F1 9.8 Tf [([17] [23] [24])] TJ ET 0.271 0.267 0.267 rg BT 443.540 526.286 Td /F1 9.8 Tf [( . Reasons for these )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(discrepancies might be caused by the use of different cellular models, species, culture conditions, )] TJ ET BT 449.478 514.381 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 478.192 514.381 Td /F1 9.8 Tf [( cultivation time, )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(experimental design or developmental differences \(e.g. age of the donor patient\) that finally result in a different myogenic )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(potential and differentiation kinetics. For instance it has been shown that differentiation kinetics of immortalized myoblast lines )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(slow down with time in culture probably due to constant selection for proliferation )] TJ ET 0.267 0.267 0.267 rg BT 376.314 478.667 Td /F1 9.8 Tf [([15])] TJ ET 0.271 0.267 0.267 rg BT 392.577 478.667 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 459.262 Td /F1 9.8 Tf [(Being able to assess new therapeutical approaches is of great significance and requires to prove the proper function of the )] TJ ET BT 26.250 447.357 Td /F1 9.8 Tf [(restored protein. This can be achieved partially by analysis of the correct intracellular localisation and the size of the protein )] TJ ET BT 26.250 435.453 Td /F1 9.8 Tf [(using immunochemical approaches. In the case of dysferlin the assumption that dysferlin is indispensable in sarcolemmal repair )] TJ ET BT 26.250 423.548 Td /F1 9.8 Tf [(opens the possibility for a direct functional assay by laser-mediated membrane wounding in cultured myotubes and myofibers. )] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(We show here that myotubes derived from the immortalized dysferlin-deficient myoblast lines, e.g. IM DYSF1 and IM DYSF2, )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(can be employed as a read-out tool of dysferlin functionality by laser-mediated wounding of the sarcolemma. Our results are in )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(accordance with the earlier observed dysfunction of the membrane resealing process in the absence of dysferlin in myotubes )] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(and myofibers )] TJ ET 0.267 0.267 0.267 rg BT 90.191 375.929 Td /F1 9.8 Tf [([6] [8] [14])] TJ ET 0.271 0.267 0.267 rg BT 133.559 375.929 Td /F1 9.8 Tf [( . We conclude that the human immortalized dysferlin-deficient myoblast lines represent innovative )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(tools to assess dysferlin functionality after application of pharmacological and genetical approaches to restore dysferlin.)] TJ ET BT 26.250 344.619 Td /F1 9.8 Tf [(Although we did not analyze cellular metabolism and regulation of cell cycle progression we expect metabolic changes in )] TJ ET BT 26.250 332.715 Td /F1 9.8 Tf [(immortalized myoblasts due to their high proliferative potential. However, this seems to have no influence on myogenic )] TJ ET BT 26.250 320.810 Td /F1 9.8 Tf [(differentiation and dysferlin function in immortalized myoblasts and their corresponding myotubes as demonstrated in this report.)] TJ ET BT 26.250 301.405 Td /F1 9.8 Tf [(In summary, the immortalized myoblast cell lines display properties highly similar to their parental cell lines with respect to )] TJ ET BT 26.250 289.500 Td /F1 9.8 Tf [(myogenic differentiation, formation of multinucleated myotubes, development of a correct myofibrillar architecture and dysferlin )] TJ ET BT 26.250 277.596 Td /F1 9.8 Tf [(protein expression. Dysferlin reveals unaltered subcellular localization and function in membrane repair in control cell lines, )] TJ ET BT 26.250 265.691 Td /F1 9.8 Tf [(while it is perturbed in cell lines derived from LGMD-2B patients. In addition dysferlin-deficient myoblasts have been described )] TJ ET BT 26.250 253.786 Td /F1 9.8 Tf [(to fully differentiate suggesting that dysferlin does not play a role in early stages of myotube formation. Therefore, immortalized )] TJ ET BT 26.250 241.881 Td /F1 9.8 Tf [(human myoblast lines harbouring different mutations in dysferlin represent a very useful tool to further investigate dysferlin )] TJ ET BT 26.250 229.977 Td /F1 9.8 Tf [(function, to study the pathophysiological mechanisms involved in dysferlinopathy and more importantly to assess therapeutic )] TJ ET BT 26.250 218.072 Td /F1 9.8 Tf [(strategies to correct dysferlinopathies with a reliable readout.)] TJ ET BT 26.250 181.469 Td /F4 12.0 Tf [(Competing interests)] TJ ET BT 26.250 161.515 Td /F1 9.8 Tf [(The authors have declared that no competing interests exist.)] TJ ET BT 26.250 124.913 Td /F4 12.0 Tf [(Address for Correspondence)] TJ ET BT 26.250 104.958 Td /F1 9.8 Tf [(Simone Spuler, MD)] TJ ET BT 26.250 85.554 Td /F1 9.8 Tf [(ECRC, Charit Campus Buch, Lindenberger Weg 80,)] TJ ET BT 26.250 66.149 Td /F1 9.8 Tf [(13125 Berlin, Germany)] TJ ET Q q 15.000 36.863 577.500 740.137 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(from them, as examplified for DYSF1 in this report. However, in the present study it was possible to get a 100% myogenic )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(population of IM DYSF1 after immortalization with hTERT and CDK-4 and subsequent cloning highlighting the great potential of )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(the immortalization procedure. We thoroughly characterized four immortalized LGMD2B patient cell lines harbouring different )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(dysferlin mutations not only for myogenic differentiation but also functionally and found no significant differences to their )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(parental cell lines \(Fig.1 and data not shown\). Therefore, the immortalization of primary human myoblasts represents a major )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(advantage and may overcome the limitations mentioned above. In addition to LGMD2B, immortalized human myoblast lines )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(have been successfully established from patients with other muscle diseases including facioscapulohumeral MD, Duchenne )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(MD, congenital MD and oculo-pharyngeal MD )] TJ ET 0.267 0.267 0.267 rg BT 226.193 684.143 Td /F1 9.8 Tf [([13] [15])] TJ ET 0.271 0.267 0.267 rg BT 261.430 684.143 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(Human cell lines with unlimited proliferative capacity are useful tools in cell biology and for translational research. Up to now, )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(only murine myoblast lines are available that are deficient for dysferlin. GREG cells were derived from A/J mice lacking dysferlin )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(expression)] TJ ET 0.267 0.267 0.267 rg BT 73.391 640.929 Td /F1 9.8 Tf [( [16] )] TJ ET 0.271 0.267 0.267 rg BT 95.075 640.929 Td /F1 9.8 Tf [(and a C2C12 cell clone with a stable dysferlin knock-down by shRNA has been establihed that expresses about )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(10% of residual dysferlin )] TJ ET 0.267 0.267 0.267 rg BT 135.167 629.024 Td /F1 9.8 Tf [([17])] TJ ET 0.271 0.267 0.267 rg BT 151.430 629.024 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 609.619 Td /F1 9.8 Tf [(There are discrepancies in the literature about the potential of dysferlin-deficient myoblasts to fuse and to differentiate into )] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(myotubes. Dysferlin-deficient myoblasts have been described to fully differentiate without any impairment as compared to wild-)] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(type myoblasts with respect to myoblast fusion and activation of myogenic pathways. This strongly suggests that dysferlin does )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(not play a role in early stages of myotube formation and subsequent maturation)] TJ ET 0.267 0.267 0.267 rg BT 368.212 573.905 Td /F1 9.8 Tf [( [7] [18] [19] [20] [21])] TJ ET 0.271 0.267 0.267 rg BT 457.658 573.905 Td /F1 9.8 Tf [( . Instead, myoferlin, )] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(another member of the ferlin protein family, has been described to be essential for myoblast-myoblast and myoblast-myotube )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(fusion )] TJ ET 0.267 0.267 0.267 rg BT 54.974 550.095 Td /F1 9.8 Tf [([18] [22])] TJ ET 0.271 0.267 0.267 rg BT 90.210 550.095 Td /F1 9.8 Tf [( . Myoferlin is expressed early during myoblast differentiation whereas dysferlin is expressed only after the )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(formation of multinucleated myotubes )] TJ ET 0.267 0.267 0.267 rg BT 190.986 538.191 Td /F1 9.8 Tf [([18] [22])] TJ ET 0.271 0.267 0.267 rg BT 226.222 538.191 Td /F1 9.8 Tf [( . Contradictorily, a reduced or delayed differentiation of dysferlin-deficient )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(myoblasts into myotubes has been attributed to dysferlin-deficiency by other groups )] TJ ET 0.267 0.267 0.267 rg BT 389.330 526.286 Td /F1 9.8 Tf [([17] [23] [24])] TJ ET 0.271 0.267 0.267 rg BT 443.540 526.286 Td /F1 9.8 Tf [( . Reasons for these )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(discrepancies might be caused by the use of different cellular models, species, culture conditions, )] TJ ET BT 449.478 514.381 Td /F5 9.8 Tf [(in vitro)] TJ ET BT 478.192 514.381 Td /F1 9.8 Tf [( cultivation time, )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(experimental design or developmental differences \(e.g. age of the donor patient\) that finally result in a different myogenic )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(potential and differentiation kinetics. For instance it has been shown that differentiation kinetics of immortalized myoblast lines )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(slow down with time in culture probably due to constant selection for proliferation )] TJ ET 0.267 0.267 0.267 rg BT 376.314 478.667 Td /F1 9.8 Tf [([15])] TJ ET 0.271 0.267 0.267 rg BT 392.577 478.667 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 459.262 Td /F1 9.8 Tf [(Being able to assess new therapeutical approaches is of great significance and requires to prove the proper function of the )] TJ ET BT 26.250 447.357 Td /F1 9.8 Tf [(restored protein. This can be achieved partially by analysis of the correct intracellular localisation and the size of the protein )] TJ ET BT 26.250 435.453 Td /F1 9.8 Tf [(using immunochemical approaches. In the case of dysferlin the assumption that dysferlin is indispensable in sarcolemmal repair )] TJ ET BT 26.250 423.548 Td /F1 9.8 Tf [(opens the possibility for a direct functional assay by laser-mediated membrane wounding in cultured myotubes and myofibers. )] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(We show here that myotubes derived from the immortalized dysferlin-deficient myoblast lines, e.g. IM DYSF1 and IM DYSF2, )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(can be employed as a read-out tool of dysferlin functionality by laser-mediated wounding of the sarcolemma. Our results are in )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(accordance with the earlier observed dysfunction of the membrane resealing process in the absence of dysferlin in myotubes )] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(and myofibers )] TJ ET 0.267 0.267 0.267 rg BT 90.191 375.929 Td /F1 9.8 Tf [([6] [8] [14])] TJ ET 0.271 0.267 0.267 rg BT 133.559 375.929 Td /F1 9.8 Tf [( . We conclude that the human immortalized dysferlin-deficient myoblast lines represent innovative )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(tools to assess dysferlin functionality after application of pharmacological and genetical approaches to restore dysferlin.)] TJ ET BT 26.250 344.619 Td /F1 9.8 Tf [(Although we did not analyze cellular metabolism and regulation of cell cycle progression we expect metabolic changes in )] TJ ET BT 26.250 332.715 Td /F1 9.8 Tf [(immortalized myoblasts due to their high proliferative potential. However, this seems to have no influence on myogenic )] TJ ET BT 26.250 320.810 Td /F1 9.8 Tf [(differentiation and dysferlin function in immortalized myoblasts and their corresponding myotubes as demonstrated in this report.)] TJ ET BT 26.250 301.405 Td /F1 9.8 Tf [(In summary, the immortalized myoblast cell lines display properties highly similar to their parental cell lines with respect to )] TJ ET BT 26.250 289.500 Td /F1 9.8 Tf [(myogenic differentiation, formation of multinucleated myotubes, development of a correct myofibrillar architecture and dysferlin )] TJ ET BT 26.250 277.596 Td /F1 9.8 Tf [(protein expression. Dysferlin reveals unaltered subcellular localization and function in membrane repair in control cell lines, )] TJ ET BT 26.250 265.691 Td /F1 9.8 Tf [(while it is perturbed in cell lines derived from LGMD-2B patients. In addition dysferlin-deficient myoblasts have been described )] TJ ET BT 26.250 253.786 Td /F1 9.8 Tf [(to fully differentiate suggesting that dysferlin does not play a role in early stages of myotube formation. Therefore, immortalized )] TJ ET BT 26.250 241.881 Td /F1 9.8 Tf [(human myoblast lines harbouring different mutations in dysferlin represent a very useful tool to further investigate dysferlin )] TJ ET BT 26.250 229.977 Td /F1 9.8 Tf [(function, to study the pathophysiological mechanisms involved in dysferlinopathy and more importantly to assess therapeutic )] TJ ET BT 26.250 218.072 Td /F1 9.8 Tf [(strategies to correct dysferlinopathies with a reliable readout.)] TJ ET BT 26.250 181.469 Td /F4 12.0 Tf [(Competing interests)] TJ ET BT 26.250 161.515 Td /F1 9.8 Tf [(The authors have declared that no competing interests exist.)] TJ ET BT 26.250 124.913 Td /F4 12.0 Tf [(Address for Correspondence)] TJ ET BT 26.250 104.958 Td /F1 9.8 Tf [(Simone Spuler, MD)] TJ ET BT 26.250 85.554 Td /F1 9.8 Tf [(ECRC, Charit Campus Buch, Lindenberger Weg 80,)] TJ ET BT 26.250 66.149 Td /F1 9.8 Tf [(13125 Berlin, Germany)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(6)] TJ ET BT 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Stephanie Meyer for expert technical assistance.)] TJ ET BT 26.250 681.817 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 654.363 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 654.363 Td /F1 9.8 Tf [(Goldstein, J. A., and McNally, E. M. \(2010\) J Gen Physiol 136, 29-34)] TJ ET BT 26.250 634.958 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 634.958 Td /F1 9.8 Tf [(Liu, J., Aoki, M., Illa, I., Wu, C., Fardeau, M., Angelini, C., Serrano, C., Urtizberea, J. A., Hentati, F., Hamida, M. B., Bohlega, )] TJ ET BT 26.250 623.053 Td /F1 9.8 Tf [(S., Culper, E. J., Amato, A. A., Bossie, K., Oeltjen, J., Bejaoui, K., McKenna-Yasek, D., Hosler, B. A., Schurr, E., Arahata, K., de )] TJ ET BT 26.250 611.148 Td /F1 9.8 Tf [(Jong, P. J., and Brown, R. H., Jr. \(1998\) Nat Genet 20, 31-36)] TJ ET BT 26.250 591.744 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 591.744 Td /F1 9.8 Tf [(Bashir, R., Britton, S., Strachan, T., Keers, S., Vafiadaki, E., Lako, M., Richard, I., Marchand, S., Bourg, N., Argov, Z., Sadeh, )] TJ ET BT 26.250 579.839 Td /F1 9.8 Tf [(M., Mahjneh, I., Marconi, G., Passos-Bueno, M. R., Moreira Ede, S., Zatz, M., Beckmann, J. S., and Bushby, K. \(1998\) Nat )] TJ ET BT 26.250 567.934 Td /F1 9.8 Tf [(Genet 20, 37-42)] TJ ET BT 26.250 548.529 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 548.529 Td /F1 9.8 Tf [(Anderson, L. V., Davison, K., Moss, J. A., Young, C., Cullen, M. J., Walsh, J., Johnson, M. A., Bashir, R., Britton, S., Keers, )] TJ ET BT 26.250 536.625 Td /F1 9.8 Tf [(S., Argov, Z., Mahjneh, I., Fougerousse, F., Beckmann, J. S., and Bushby, K. M. \(1999\) Hum Mol Genet 8, 855-861)] TJ ET BT 26.250 517.220 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 517.220 Td /F1 9.8 Tf [(Matsuda, C., Hayashi, Y. K., Ogawa, M., Aoki, M., Murayama, K., Nishino, I., Nonaka, I., Arahata, K., and Brown, R. H., Jr. )] TJ ET BT 26.250 505.315 Td /F1 9.8 Tf [(\(2001\) Hum Mol Genet 10, 1761-1766)] TJ ET BT 26.250 485.910 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 485.910 Td /F1 9.8 Tf [(Lennon, N. J., Kho, A., Bacskai, B. J., Perlmutter, S. L., Hyman, B. T., and Brown, R. H., Jr. \(2003\) J Biol Chem 278, 50466-)] TJ ET BT 26.250 474.006 Td /F1 9.8 Tf [(50473)] TJ ET BT 26.250 454.601 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 454.601 Td /F1 9.8 Tf [(Chiu, Y. H., Hornsey, M. A., Klinge, L., Jorgensen, L. H., Laval, S. H., Charlton, R., Barresi, R., Straub, V., Lochmuller, H., )] TJ ET BT 26.250 442.696 Td /F1 9.8 Tf [(and Bushby, K. \(2009\) Hum Mol Genet 18, 1976-1989)] TJ ET BT 26.250 423.291 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 423.291 Td /F1 9.8 Tf [(Bansal, D., Miyake, K., Vogel, S. S., Groh, S., Chen, C. C., Williamson, R., McNeil, P. L., and Campbell, K. P. \(2003\) Nature )] TJ ET BT 26.250 411.387 Td /F1 9.8 Tf [(423, 168-172)] TJ ET BT 26.250 391.982 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 391.982 Td /F1 9.8 Tf [(Krahn, M., Beroud, C., Labelle, V., Nguyen, K., Bernard, R., Bassez, G., Figarella-Branger, D., Fernandez, C., Bouvenot, J., )] TJ ET BT 26.250 380.077 Td /F1 9.8 Tf [(Richard, I., Ollagnon-Roman, E., Bevilacqua, J. A., Salvo, E., Attarian, S., Chapon, F., Pellissier, J. F., Pouget, J., Hammouda )] TJ ET BT 26.250 368.172 Td /F1 9.8 Tf [(el, H., Laforet, P., Urtizberea, J. A., Eymard, B., Leturcq, F., and Levy, N. \(2009\) Hum Mutat 30, E345-375)] TJ ET BT 26.250 348.768 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 348.768 Td /F1 9.8 Tf [(Spuler, S., Carl, M., Zabojszcza, J., Straub, V., Bushby, K., Moore, S. A., Bahring, S., Wenzel, K., Vinkemeier, U., and )] TJ ET BT 26.250 336.863 Td /F1 9.8 Tf [(Rocken, C. \(2008\) Ann Neurol 63, 323-328)] TJ ET BT 26.250 317.458 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 317.458 Td /F1 9.8 Tf [(Wenzel, K., Carl, M., Perrot, A., Zabojszcza, J., Assadi, M., Ebeling, M., Geier, C., Robinson, P. N., Kress, W., Osterziel, K. )] TJ ET BT 26.250 305.553 Td /F1 9.8 Tf [(J., and Spuler, S. \(2006\) Hum Mutat 27, 599-600)] TJ ET BT 26.250 286.149 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 286.149 Td /F1 9.8 Tf [(Bigot, A., Jacquemin, V., Debacq-Chainiaux, F., Butler-Browne, G. S., Toussaint, O., Furling, D., and Mouly, V. \(2008\) Biol )] TJ ET BT 26.250 274.244 Td /F1 9.8 Tf [(Cell 100, 189-199)] TJ ET BT 26.250 254.839 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 254.839 Td /F1 9.8 Tf [(Mamchaoui, K., Trollet, C., Bigot, A., Negroni, E., Chaouch, S., Wolff, A., Kandalla, P. K., Marie, S., Di Santo, J., Lacau St )] TJ ET BT 26.250 242.934 Td /F1 9.8 Tf [(Guily, J., Muntoni, F., Kim, J., Philippi, S., Spuler, S., Levy, N., Blumen, S. C., Voit, T., Wright, W. E., Aamiri, A., Butler-Browne, )] TJ ET BT 26.250 231.030 Td /F1 9.8 Tf [(G. S., and Mouly, V. \(2011\) Skelet Muscle 1, 34)] TJ ET BT 26.250 211.625 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 211.625 Td /F1 9.8 Tf [(Cai, C., Masumiya, H., Weisleder, N., Matsuda, N., Nishi, M., Hwang, M., Ko, J. K., Lin, P., Thornton, A., Zhao, X., Pan, Z., )] TJ ET BT 26.250 199.720 Td /F1 9.8 Tf [(Komazaki, S., Brotto, M., Takeshima, H., and Ma, J. \(2009\) Nat Cell Biol 11, 56-64)] TJ ET BT 26.250 180.315 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 180.315 Td /F1 9.8 Tf [(Stadler, G., Chen, J. C., Wagner, K., Robin, J. D., Shay, J. W., Emerson, C. P., Jr., and Wright, W. E. \(2011\) Skelet Muscle )] TJ ET BT 26.250 168.411 Td /F1 9.8 Tf [(1, 12)] TJ ET BT 26.250 149.006 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 149.006 Td /F1 9.8 Tf [(Humphrey, G. W., Mekhedov, E., Blank, P. S., de Morree, A., Pekkurnaz, G., Nagaraju, K., and Zimmerberg, J. \(2012\) Exp )] TJ ET BT 26.250 137.101 Td /F1 9.8 Tf [(Cell Res 318, 127-135)] TJ ET BT 26.250 117.696 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 117.696 Td /F1 9.8 Tf [(Belanto, J. J., Diaz-Perez, S. V., Magyar, C. E., Maxwell, M. M., Yilmaz, Y., Topp, K., Boso, G., Jamieson, C. H., Cacalano, )] TJ ET BT 26.250 105.792 Td /F1 9.8 Tf [(N. A., and Jamieson, C. A. \(2010\) Neuromuscul Disord 20, 111-121)] TJ ET BT 26.250 86.387 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 86.387 Td /F1 9.8 Tf [(Doherty, K. R., Cave, A., Davis, D. B., Delmonte, A. J., Posey, A., Earley, J. U., Hadhazy, M., and McNally, E. M. \(2005\) )] TJ ET BT 26.250 74.482 Td /F1 9.8 Tf [(Development 132, 5565-5575)] TJ ET BT 26.250 55.077 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 55.077 Td /F1 9.8 Tf [(Klinge, L., Laval, S., Keers, S., Haldane, F., Straub, V., Barresi, R., and Bushby, K. \(2007\) Faseb J 21, 1768-1776)] TJ ET Q q 15.000 45.197 577.500 731.803 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(T+49 30 450 540501, F: +49 30 450 540906, e-mail: simone.spuler@charite.de)] TJ ET BT 26.250 730.874 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 710.919 Td /F1 9.8 Tf [(We thank Stephanie Meyer for expert technical assistance.)] TJ ET BT 26.250 681.817 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 654.363 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 654.363 Td /F1 9.8 Tf [(Goldstein, J. A., and McNally, E. M. \(2010\) J Gen Physiol 136, 29-34)] TJ ET BT 26.250 634.958 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 634.958 Td /F1 9.8 Tf [(Liu, J., Aoki, M., Illa, I., Wu, C., Fardeau, M., Angelini, C., Serrano, C., Urtizberea, J. A., Hentati, F., Hamida, M. B., Bohlega, )] TJ ET BT 26.250 623.053 Td /F1 9.8 Tf [(S., Culper, E. J., Amato, A. A., Bossie, K., Oeltjen, J., Bejaoui, K., McKenna-Yasek, D., Hosler, B. A., Schurr, E., Arahata, K., de )] TJ ET BT 26.250 611.148 Td /F1 9.8 Tf [(Jong, P. J., and Brown, R. H., Jr. \(1998\) Nat Genet 20, 31-36)] TJ ET BT 26.250 591.744 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 591.744 Td /F1 9.8 Tf [(Bashir, R., Britton, S., Strachan, T., Keers, S., Vafiadaki, E., Lako, M., Richard, I., Marchand, S., Bourg, N., Argov, Z., Sadeh, )] TJ ET BT 26.250 579.839 Td /F1 9.8 Tf [(M., Mahjneh, I., Marconi, G., Passos-Bueno, M. R., Moreira Ede, S., Zatz, M., Beckmann, J. S., and Bushby, K. \(1998\) Nat )] TJ ET BT 26.250 567.934 Td /F1 9.8 Tf [(Genet 20, 37-42)] TJ ET BT 26.250 548.529 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 548.529 Td /F1 9.8 Tf [(Anderson, L. V., Davison, K., Moss, J. A., Young, C., Cullen, M. J., Walsh, J., Johnson, M. A., Bashir, R., Britton, S., Keers, )] TJ ET BT 26.250 536.625 Td /F1 9.8 Tf [(S., Argov, Z., Mahjneh, I., Fougerousse, F., Beckmann, J. S., and Bushby, K. M. \(1999\) Hum Mol Genet 8, 855-861)] TJ ET BT 26.250 517.220 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 517.220 Td /F1 9.8 Tf [(Matsuda, C., Hayashi, Y. K., Ogawa, M., Aoki, M., Murayama, K., Nishino, I., Nonaka, I., Arahata, K., and Brown, R. H., Jr. )] TJ ET BT 26.250 505.315 Td /F1 9.8 Tf [(\(2001\) Hum Mol Genet 10, 1761-1766)] TJ ET BT 26.250 485.910 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 485.910 Td /F1 9.8 Tf [(Lennon, N. J., Kho, A., Bacskai, B. J., Perlmutter, S. L., Hyman, B. T., and Brown, R. H., Jr. \(2003\) J Biol Chem 278, 50466-)] TJ ET BT 26.250 474.006 Td /F1 9.8 Tf [(50473)] TJ ET BT 26.250 454.601 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 454.601 Td /F1 9.8 Tf [(Chiu, Y. H., Hornsey, M. A., Klinge, L., Jorgensen, L. H., Laval, S. H., Charlton, R., Barresi, R., Straub, V., Lochmuller, H., )] TJ ET BT 26.250 442.696 Td /F1 9.8 Tf [(and Bushby, K. \(2009\) Hum Mol Genet 18, 1976-1989)] TJ ET BT 26.250 423.291 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 423.291 Td /F1 9.8 Tf [(Bansal, D., Miyake, K., Vogel, S. S., Groh, S., Chen, C. C., Williamson, R., McNeil, P. L., and Campbell, K. P. \(2003\) Nature )] TJ ET BT 26.250 411.387 Td /F1 9.8 Tf [(423, 168-172)] TJ ET BT 26.250 391.982 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 391.982 Td /F1 9.8 Tf [(Krahn, M., Beroud, C., Labelle, V., Nguyen, K., Bernard, R., Bassez, G., Figarella-Branger, D., Fernandez, C., Bouvenot, J., )] TJ ET BT 26.250 380.077 Td /F1 9.8 Tf [(Richard, I., Ollagnon-Roman, E., Bevilacqua, J. A., Salvo, E., Attarian, S., Chapon, F., Pellissier, J. F., Pouget, J., Hammouda )] TJ ET BT 26.250 368.172 Td /F1 9.8 Tf [(el, H., Laforet, P., Urtizberea, J. A., Eymard, B., Leturcq, F., and Levy, N. \(2009\) Hum Mutat 30, E345-375)] TJ ET BT 26.250 348.768 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 348.768 Td /F1 9.8 Tf [(Spuler, S., Carl, M., Zabojszcza, J., Straub, V., Bushby, K., Moore, S. A., Bahring, S., Wenzel, K., Vinkemeier, U., and )] TJ ET BT 26.250 336.863 Td /F1 9.8 Tf [(Rocken, C. \(2008\) Ann Neurol 63, 323-328)] TJ ET BT 26.250 317.458 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 317.458 Td /F1 9.8 Tf [(Wenzel, K., Carl, M., Perrot, A., Zabojszcza, J., Assadi, M., Ebeling, M., Geier, C., Robinson, P. N., Kress, W., Osterziel, K. )] TJ ET BT 26.250 305.553 Td /F1 9.8 Tf [(J., and Spuler, S. \(2006\) Hum Mutat 27, 599-600)] TJ ET BT 26.250 286.149 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 286.149 Td /F1 9.8 Tf [(Bigot, A., Jacquemin, V., Debacq-Chainiaux, F., Butler-Browne, G. S., Toussaint, O., Furling, D., and Mouly, V. \(2008\) Biol )] TJ ET BT 26.250 274.244 Td /F1 9.8 Tf [(Cell 100, 189-199)] TJ ET BT 26.250 254.839 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 254.839 Td /F1 9.8 Tf [(Mamchaoui, K., Trollet, C., Bigot, A., Negroni, E., Chaouch, S., Wolff, A., Kandalla, P. K., Marie, S., Di Santo, J., Lacau St )] TJ ET BT 26.250 242.934 Td /F1 9.8 Tf [(Guily, J., Muntoni, F., Kim, J., Philippi, S., Spuler, S., Levy, N., Blumen, S. C., Voit, T., Wright, W. E., Aamiri, A., Butler-Browne, )] TJ ET BT 26.250 231.030 Td /F1 9.8 Tf [(G. S., and Mouly, V. \(2011\) Skelet Muscle 1, 34)] TJ ET BT 26.250 211.625 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 211.625 Td /F1 9.8 Tf [(Cai, C., Masumiya, H., Weisleder, N., Matsuda, N., Nishi, M., Hwang, M., Ko, J. K., Lin, P., Thornton, A., Zhao, X., Pan, Z., )] TJ ET BT 26.250 199.720 Td /F1 9.8 Tf [(Komazaki, S., Brotto, M., Takeshima, H., and Ma, J. \(2009\) Nat Cell Biol 11, 56-64)] TJ ET BT 26.250 180.315 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 180.315 Td /F1 9.8 Tf [(Stadler, G., Chen, J. C., Wagner, K., Robin, J. D., Shay, J. W., Emerson, C. P., Jr., and Wright, W. E. \(2011\) Skelet Muscle )] TJ ET BT 26.250 168.411 Td /F1 9.8 Tf [(1, 12)] TJ ET BT 26.250 149.006 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 149.006 Td /F1 9.8 Tf [(Humphrey, G. W., Mekhedov, E., Blank, P. S., de Morree, A., Pekkurnaz, G., Nagaraju, K., and Zimmerberg, J. \(2012\) Exp )] TJ ET BT 26.250 137.101 Td /F1 9.8 Tf [(Cell Res 318, 127-135)] TJ ET BT 26.250 117.696 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 117.696 Td /F1 9.8 Tf [(Belanto, J. J., Diaz-Perez, S. V., Magyar, C. E., Maxwell, M. M., Yilmaz, Y., Topp, K., Boso, G., Jamieson, C. H., Cacalano, )] TJ ET BT 26.250 105.792 Td /F1 9.8 Tf [(N. A., and Jamieson, C. A. \(2010\) Neuromuscul Disord 20, 111-121)] TJ ET BT 26.250 86.387 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 86.387 Td /F1 9.8 Tf [(Doherty, K. R., Cave, A., Davis, D. B., Delmonte, A. J., Posey, A., Earley, J. U., Hadhazy, M., and McNally, E. M. \(2005\) )] TJ ET BT 26.250 74.482 Td /F1 9.8 Tf [(Development 132, 5565-5575)] TJ ET BT 26.250 55.077 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 55.077 Td /F1 9.8 Tf [(Klinge, L., Laval, S., Keers, S., Haldane, F., Straub, V., Barresi, R., and Bushby, K. \(2007\) Faseb J 21, 1768-1776)] TJ ET Q q 15.000 45.197 577.500 731.803 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(T+49 30 450 540501, F: +49 30 450 540906, e-mail: simone.spuler@charite.de)] TJ ET BT 26.250 730.874 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 710.919 Td /F1 9.8 Tf [(We thank Stephanie Meyer for expert technical assistance.)] TJ ET BT 26.250 681.817 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 654.363 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 654.363 Td /F1 9.8 Tf [(Goldstein, J. A., and McNally, E. 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H., Jr. \(2003\) J Biol Chem 278, 50466-)] TJ ET BT 26.250 474.006 Td /F1 9.8 Tf [(50473)] TJ ET BT 26.250 454.601 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 454.601 Td /F1 9.8 Tf [(Chiu, Y. H., Hornsey, M. A., Klinge, L., Jorgensen, L. H., Laval, S. H., Charlton, R., Barresi, R., Straub, V., Lochmuller, H., )] TJ ET BT 26.250 442.696 Td /F1 9.8 Tf [(and Bushby, K. \(2009\) Hum Mol Genet 18, 1976-1989)] TJ ET BT 26.250 423.291 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 423.291 Td /F1 9.8 Tf [(Bansal, D., Miyake, K., Vogel, S. S., Groh, S., Chen, C. C., Williamson, R., McNeil, P. L., and Campbell, K. P. \(2003\) Nature )] TJ ET BT 26.250 411.387 Td /F1 9.8 Tf [(423, 168-172)] TJ ET BT 26.250 391.982 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 391.982 Td /F1 9.8 Tf [(Krahn, M., Beroud, C., Labelle, V., Nguyen, K., Bernard, R., Bassez, G., Figarella-Branger, D., Fernandez, C., Bouvenot, J., )] TJ ET BT 26.250 380.077 Td /F1 9.8 Tf [(Richard, I., Ollagnon-Roman, E., Bevilacqua, J. A., Salvo, E., Attarian, S., Chapon, F., Pellissier, J. F., Pouget, J., Hammouda )] TJ ET BT 26.250 368.172 Td /F1 9.8 Tf [(el, H., Laforet, P., Urtizberea, J. A., Eymard, B., Leturcq, F., and Levy, N. \(2009\) Hum Mutat 30, E345-375)] TJ ET BT 26.250 348.768 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 348.768 Td /F1 9.8 Tf [(Spuler, S., Carl, M., Zabojszcza, J., Straub, V., Bushby, K., Moore, S. A., Bahring, S., Wenzel, K., Vinkemeier, U., and )] TJ ET BT 26.250 336.863 Td /F1 9.8 Tf [(Rocken, C. \(2008\) Ann Neurol 63, 323-328)] TJ ET BT 26.250 317.458 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 317.458 Td /F1 9.8 Tf [(Wenzel, K., Carl, M., Perrot, A., Zabojszcza, J., Assadi, M., Ebeling, M., Geier, C., Robinson, P. N., Kress, W., Osterziel, K. )] TJ ET BT 26.250 305.553 Td /F1 9.8 Tf [(J., and Spuler, S. \(2006\) Hum Mutat 27, 599-600)] TJ ET BT 26.250 286.149 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 286.149 Td /F1 9.8 Tf [(Bigot, A., Jacquemin, V., Debacq-Chainiaux, F., Butler-Browne, G. S., Toussaint, O., Furling, D., and Mouly, V. \(2008\) Biol )] TJ ET BT 26.250 274.244 Td /F1 9.8 Tf [(Cell 100, 189-199)] TJ ET BT 26.250 254.839 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 254.839 Td /F1 9.8 Tf [(Mamchaoui, K., Trollet, C., Bigot, A., Negroni, E., Chaouch, S., Wolff, A., Kandalla, P. K., Marie, S., Di Santo, J., Lacau St )] TJ ET BT 26.250 242.934 Td /F1 9.8 Tf [(Guily, J., Muntoni, F., Kim, J., Philippi, S., Spuler, S., Levy, N., Blumen, S. C., Voit, T., Wright, W. E., Aamiri, A., Butler-Browne, )] TJ ET BT 26.250 231.030 Td /F1 9.8 Tf [(G. S., and Mouly, V. \(2011\) Skelet Muscle 1, 34)] TJ ET BT 26.250 211.625 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 211.625 Td /F1 9.8 Tf [(Cai, C., Masumiya, H., Weisleder, N., Matsuda, N., Nishi, M., Hwang, M., Ko, J. K., Lin, P., Thornton, A., Zhao, X., Pan, Z., )] TJ ET BT 26.250 199.720 Td /F1 9.8 Tf [(Komazaki, S., Brotto, M., Takeshima, H., and Ma, J. \(2009\) Nat Cell Biol 11, 56-64)] TJ ET BT 26.250 180.315 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 180.315 Td /F1 9.8 Tf [(Stadler, G., Chen, J. C., Wagner, K., Robin, J. D., Shay, J. W., Emerson, C. P., Jr., and Wright, W. E. \(2011\) Skelet Muscle )] TJ ET BT 26.250 168.411 Td /F1 9.8 Tf [(1, 12)] TJ ET BT 26.250 149.006 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 149.006 Td /F1 9.8 Tf [(Humphrey, G. W., Mekhedov, E., Blank, P. S., de Morree, A., Pekkurnaz, G., Nagaraju, K., and Zimmerberg, J. \(2012\) Exp )] TJ ET BT 26.250 137.101 Td /F1 9.8 Tf [(Cell Res 318, 127-135)] TJ ET BT 26.250 117.696 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 117.696 Td /F1 9.8 Tf [(Belanto, J. J., Diaz-Perez, S. V., Magyar, C. E., Maxwell, M. M., Yilmaz, Y., Topp, K., Boso, G., Jamieson, C. H., Cacalano, )] TJ ET BT 26.250 105.792 Td /F1 9.8 Tf [(N. A., and Jamieson, C. A. \(2010\) Neuromuscul Disord 20, 111-121)] TJ ET BT 26.250 86.387 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 86.387 Td /F1 9.8 Tf [(Doherty, K. R., Cave, A., Davis, D. B., Delmonte, A. J., Posey, A., Earley, J. U., Hadhazy, M., and McNally, E. M. \(2005\) )] TJ ET BT 26.250 74.482 Td /F1 9.8 Tf [(Development 132, 5565-5575)] TJ ET BT 26.250 55.077 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 55.077 Td /F1 9.8 Tf [(Klinge, L., Laval, S., Keers, S., Haldane, F., Straub, V., Barresi, R., and Bushby, K. \(2007\) Faseb J 21, 1768-1776)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(7)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Muscular Dystrophy)] TJ ET Q endstream endobj 235 0 obj << /Type /Page /Parent 3 0 R /Contents 236 0 R >> endobj 236 0 obj << /Length 4069 >> stream 0.271 0.267 0.267 rg q 15.000 629.262 577.500 147.738 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Posey, A. D., Jr., Demonbreun, A., and McNally, E. M. \(2011\) Curr Top Dev Biol 96, 203-230)] TJ ET BT 26.250 740.571 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 740.571 Td /F1 9.8 Tf [(Davis, D. B., Doherty, K. R., Delmonte, A. J., and McNally, E. M. \(2002\) J Biol Chem 277, 22883-22888)] TJ ET BT 26.250 721.167 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 721.167 Td /F1 9.8 Tf [(Doherty, K. R., Demonbreun, A. R., Wallace, G. Q., Cave, A., Posey, A. D., Heretis, K., Pytel, P., and McNally, E. M. \(2008\) )] TJ ET BT 26.250 709.262 Td /F1 9.8 Tf [(J Biol Chem 283, 20252-20260)] TJ ET BT 26.250 689.857 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 689.857 Td /F1 9.8 Tf [(de Luna, N., Gallardo, E., Soriano, M., Dominguez-Perles, R., de la Torre, C., Rojas-Garcia, R., Garcia-Verdugo, J. M., and )] TJ ET BT 26.250 677.952 Td /F1 9.8 Tf [(Illa, I. \(2006\) J Biol Chem 281, 17092-17098)] TJ ET BT 26.250 658.548 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 658.548 Td /F1 9.8 Tf [(Demonbreun, A. R., Fahrenbach, J. P., Deveaux, K., Earley, J. U., Pytel, P., and McNally, E. M. \(2011\) Hum Mol Genet 20, )] TJ ET BT 26.250 646.643 Td /F1 9.8 Tf [(779-789)] TJ ET Q q 15.000 629.262 577.500 147.738 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Posey, A. D., Jr., Demonbreun, A., and McNally, E. M. \(2011\) Curr Top Dev Biol 96, 203-230)] TJ ET BT 26.250 740.571 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 740.571 Td /F1 9.8 Tf [(Davis, D. B., Doherty, K. R., Delmonte, A. J., and McNally, E. M. \(2002\) J Biol Chem 277, 22883-22888)] TJ ET BT 26.250 721.167 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 721.167 Td /F1 9.8 Tf [(Doherty, K. R., Demonbreun, A. R., Wallace, G. Q., Cave, A., Posey, A. D., Heretis, K., Pytel, P., and McNally, E. M. \(2008\) )] TJ ET BT 26.250 709.262 Td /F1 9.8 Tf [(J Biol Chem 283, 20252-20260)] TJ ET BT 26.250 689.857 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 689.857 Td /F1 9.8 Tf [(de Luna, N., Gallardo, E., Soriano, M., Dominguez-Perles, R., de la Torre, C., Rojas-Garcia, R., Garcia-Verdugo, J. M., and )] TJ ET BT 26.250 677.952 Td /F1 9.8 Tf [(Illa, I. \(2006\) J Biol Chem 281, 17092-17098)] TJ ET BT 26.250 658.548 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 658.548 Td /F1 9.8 Tf [(Demonbreun, A. R., Fahrenbach, J. P., Deveaux, K., Earley, J. U., Pytel, P., and McNally, E. M. \(2011\) Hum Mol Genet 20, )] TJ ET BT 26.250 646.643 Td /F1 9.8 Tf [(779-789)] TJ ET Q q 15.000 629.262 577.500 147.738 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Posey, A. D., Jr., Demonbreun, A., and McNally, E. M. \(2011\) Curr Top Dev Biol 96, 203-230)] TJ ET BT 26.250 740.571 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 740.571 Td /F1 9.8 Tf [(Davis, D. B., Doherty, K. R., Delmonte, A. J., and McNally, E. M. \(2002\) J Biol Chem 277, 22883-22888)] TJ ET BT 26.250 721.167 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 721.167 Td /F1 9.8 Tf [(Doherty, K. R., Demonbreun, A. R., Wallace, G. Q., Cave, A., Posey, A. D., Heretis, K., Pytel, P., and McNally, E. M. \(2008\) )] TJ ET BT 26.250 709.262 Td /F1 9.8 Tf [(J Biol Chem 283, 20252-20260)] TJ ET BT 26.250 689.857 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 689.857 Td /F1 9.8 Tf [(de Luna, N., Gallardo, E., Soriano, M., Dominguez-Perles, R., de la Torre, C., Rojas-Garcia, R., Garcia-Verdugo, J. M., and )] TJ ET BT 26.250 677.952 Td /F1 9.8 Tf [(Illa, I. \(2006\) J Biol Chem 281, 17092-17098)] TJ ET BT 26.250 658.548 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 658.548 Td /F1 9.8 Tf [(Demonbreun, A. R., Fahrenbach, J. P., Deveaux, K., Earley, J. U., Pytel, P., and McNally, E. 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