Muscle Explant-Derived Myoblast Isolation and Differentiation

Total muscle explant-derived cells were purified by Magnetic-activated cell sorting (Milteny Biotech) using anti-CD56 antibody (Table S1). Myoblast identity was determined by desmin immunostaining (> 98%). The primary and immortalized myoblasts were grown, respectively, in DMEM with high glucose and l-glutamine (Lonza), 10% fetal bovine serum (FBS) (Invitrogen), 1% Ultroser G (Pall BioSepra, Cergy-St-Christophe, France), and gentamicin (50 μg/ml, Sigma-Aldrich) or DMEM high glucose supplemented with 16.5% medium 199 (Lonza), 15% FBS, Ultroser G, HEPES 1 M (Sigma-Aldrich), zinc sulfate (Sigma ®-Aldrich, vitamin B12 (Sigma-Aldrich), and penicillin/streptomycin (pen/strep) at 37 °C under atmosphere with 5% CO₂. For myogenic differentiation, cells were cultured on Matrigel-coated culture dishes and a differentiation medium was added after cells reached 100% confluence. This medium was composed of DMEM/gentamicin (50 μg/ml) with 2% FBS for primary cells and DMEM high glucose, medium 199 supplemented with 0.5% insulin, 1% apo-transferrin (Sigma-Aldrich), 2% HEPES 1 M and pen/strep for immortalized cells. HEK293 were grown in DMEM high glucose-10% FBS and pen/strep. Transfection of primary cells was previously reported [3 ]. The KLF15 expression vector was a generous gift of Prof. Yegor Vassetzky [18 (link)].