C2C12 cell culture and differentiation

Myoblasts derived from mouse skeletal muscle (cat. no. CRL-1772; American Type Culture Collection) were cultured in 6-well plates containing Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere of 5% CO₂. When myoblasts were approximately 80–90% confluent, myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 2% FBS. Differentiation was allowed to proceed for 6 days, with the medium changed every 48 h, at which stage 90–100% of the cells had fused into myotubes.