Molecular Regulation of Myoblast Differentiation

C2C12 myoblasts described previously (4 (link)) or mouse primary myoblasts were maintained and induced to differentiate into myotubes, and they were treated with STO-609 (Calbiochem), EGTA (Wako), GsMTx-4 (Peptide Institute), Yoda1, nifedipine (Wako), or tranilast (Tokyo Chemical Industry). Adenoviral vectors for LacZ and mouse KLF15 were described previously (35 (link)). Mouse KLF15 and negative control siRNAs were obtained from Invitrogen and were delivered into cells with the use of the Lipofectamine RNAiMAX transfection reagent (Invitrogen). Isolation of total RNA and quantitative RT-PCR analysis were performed as previously described (4 (link)). Data were normalized by the amount of 36B4 mRNA. The sequences of PCR primers are provided in Supplemental Tables 2 and 3. Immunoblot analysis was performed with antibodies against STAT3 (4904, Cell Signaling Technology) and against Tyr⁷⁰⁵-phosphorylated STAT3 (9131, Cell Signaling Technology). Uncropped immunoblots are presented in Supplemental Figure 12.