C2C12 myoblasts were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum, 15% calf serum, penicillin (100 IU/ml), and streptomycin (100 μg/ml) (Invitrogen, Carlsbad, CA). Differentiation of myoblasts into myotubes was performed by incubating 100% confluent myoblast with differentiating medium [2% horse serum, penicillin (100 IU/ml), and streptomycin (100 μg/ml)] for 4 days. C2C12 differentiation was confirmed by morphological changes as previously described (64).

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