Myogenic Differentiation of C2C12 and Primary Myoblasts

A mouse myoblast cell line, C2C12, was cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C under 5% CO₂. Mouse primary myoblasts were isolated from lower extremity muscles of 8-week-old C57BL6J mice as described previously [48 (link)]. Myogenic differentiation of C2C12 cells and primary myoblasts were induced by replacing the medium with the differentiation medium, DMEM supplemented with 2% or 5% horse serum, respectively. Cells were transfected with 50 nM of Stealth RNAi (Thermo Fisher Scientific, Waltham, MA, USA) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) or RNAiMax (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The following siRNAs were used: Stealth RNAi siRNA negative control (negative control, Med GC, Thermo Fisher Scientific, Waltham, MA, USA), Stealth RNAi for Myoparr, and Stealth RNAi siRNAs specific for hnRNPK (MSS205172 and MSS205173, Thermo Fisher Scientific, Waltham, MA, USA). The siRNA sequences are listed in Table S1. At 24 h after siRNA transfection, myogenic differentiation was induced. At 24 h or 72 h after differentiation induction, cells were collected for the analysis of RNAs and proteins. For ISRIB treatment, the differentiation medium was added either with or without 1 µM trans-ISRIB (No.16258, Cayman Chemical Company, Ann Arbor, MI, USA).