Myoblast Differentiation and Silencing

Mouse C2C12 myoblasts were cultured in growth medium (Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies) containing 20% fetal bovine serum (FBS, Gibco) and antibiotics. Human KM155 myoblasts were cultured in growth medium (equal volume mixture of Hamm’s F10 media with 20% FBS and Promocell Skeletal Muscle Cell Growth Medium). The C2C12 and KM155 myoblasts were maintained at 37°C in a 5% CO₂ humidified atmosphere and were differentiated by replacing the growth medium with differentiation medium (DMEM with 2% horse serum) [6 (link)]. For silencing experiments, control small interfering RNA (Ctrl siRNA) or SPARC siRNA was transfected twice, 36 h and 12 h before inducing differentiation. MicroRNA mimics and inhibitors for miR-451a and miR-144-3p (Sigma Aldrich) were transfected twice, 36 h and 12 h before induction of differentiation at a final concentration of 50 nM using Lipofectamine 2000 (Life Technologies). Creatine kinase (CK) activity was determined in cell lysates using the EnzyChrom creatine kinase assay kit (BioAssay Systems) following the manufacturer's protocol.