Muscle Fiber Isolation and Culture

EDL muscles were removed carefully and digested with 2 mg ml⁻¹ collagenase type 1 (Worthington) in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) for 45 min⁻¹ h at 37 °C. Digestion was stopped by carefully transferring EDL muscles to a horse serum-coated Petri dish (60-mm) with DMEM. Myofibers were released by gently flushing muscles with a large bore glass pipette. Released single myofiber was either fixed immediately with 4% PFA or transferred and cultured in a horse serum-coated Petri dish (60-mm) in DMEM supplemented with 20% fetal bovine serum (FBS, Gibco), 4 ng ml⁻¹ basic fibroblast growth factor (bFgf, Peprotech), and 1% penicillin–streptomycin (P/S, Gibco) at 37 °C for indicated days following the protocol by Yue and colleagues^{46 (link)}. Dox (RPI) was administered in culture medium at a concentration of 1 µg ml⁻¹ to induce OSKM expression. Recombinant Wnt4 (R&D systems) was administered in culture medium at a concentration of 100 ng ml⁻¹ according to a previous study²³ .