C2c12 cells

C2C12 cells (#CRL-1772, American Type Culture Collection, ATCC, Manassas, VA, United States) were cultured in DMEM (Corning, Cultek, Spain, #10–013) supplemented with 10% FBS (Gibco, #11573397) and 1% penicillin–streptomycin (Sigma, #P4333). To induce myogenic differentiation, 70% confluence C2C12 cells were cultured in DMEM containing 2% HS (Gibco, #11540636) (Wong et al., 2020 (link)). Cells were maintained in a humidified chamber at 37°C, in 95% air and 5% CO₂. Media were replaced with fresh media every 48 h.

Primary MuSCs were isolated as previously described.²⁴ Briefly, TA muscles from 3‐month‐old mice were dissected and dissociated with collagenase (Roche, Indianapolis, IN, USA). The cells were negatively selected by biotinylated CD45, CD11, CD31 and Sca1 antibodies. The muscle cells in the flowthrough were subjected to CD34‐FITC (BD biosciences) and integrin α₇‐allophycocyanin (R&D systems, Minneapolis, MN, USA) staining. The viable PI⁻CD34⁺integrin‐α₇⁺ MuSCs were collected by FACS sorting (Influx, BD biosciences, Franklin Lake, NJ, USA). MuSCs were cultured on collagen coated dishes in F10 medium containing 10% foetal bovine serum (FBS), 5 ng/mL IL‐1α, 5 ng/mL IL‐13, 10 ng/mL IFN‐γ, and 10 ng/mL TNF‐α (R&D Systems), and 2.5 ng/mL FGF (Invitrogen, Red Wood City, CA, USA) as described previously.²⁴ MuSCs were differentiated in differentiation medium [DMEM medium (invitrogen)] containing 2% horse serum (HyClone, Malborough, MA, USA) for 3 days. C2C12 cells (ATCC) were cultured in DMEM medium (Invitrogen) containing 10% FBS (HyClone) and differentiated in DMEM medium (Invitrogen) containing 2% horse serum (HyClone) for 3 days. The differentiated myotubes were further isolated by pre‐attaching to plates for three times.

Primary myoblasts were isolated as previously described^{58 (link)}. The primary myoblasts were cultured in collagen-coated dishes in F10 basal medium (Invitrogen) containing 10% FBS and 2.5 ng/ml FGF (Invitrogen). The cells were then differentiated in differentiation medium (DMEM medium (invitrogen) containing 2% horse serum (HyClone)) for 2 days. C2C12 cells (ATCC) were cultured in DMEM medium (Invitrogen) containing 10% FBS (HyClone), and differentiated in DMEM medium (Invitrogen) containing 2% horse serum (HyClone) for 3 days. The differentiated myotubes were further isolated by pre-attaching to plates for three times. horse serum was removed from the differentiation medium when serum starvation was performed.

The C2C12 cells were purchased from Anweisci (Shanghai, China). The cells were cultured in a complete medium containing 89% DMEM (Gibco, Waltham, MA, USA), 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco, Waltham, MA, USA) and cultured in a 37 °C incubator containing 5% CO₂. For myogenic differentiation, the complete medium was changed into DMEM containing 2% horse serum (Hyclone, Logan, UT, USA). Three siRNAs, Si-NC, PCDNA3.1-MYL4 and PCDNA3.1, were purchased from JTS Scientific (Wuhan, China); the sequences were as follows (Table 1). The C2C12 cells were seeded into 6-well plates after reaching 80% confluence, and Si-MYL4, Si-NC, PCDNA3.1-MYL4 and PCDNA3.1 were transfected into the cells using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) after reaching 50% confluence.

C2C12 cells were obtained from American Type Culture Collection (ATCC, CRL-1772™, Manassas, VA, USA). The cells were cultured in growth medium consisting of Dulbecco's modified eagle medium (DME-M), 10% heat-inactivated fetal calf serum (Biowest, St. Louis, MO), and 1% penicillin-streptomycin. C2C12 cells were successfully differentiated into myocytes or myotubes in a differentiation medium consisting of DMEM, 2% heat-inactivated horse serum (Invitrogen, Carlsbad, CA, USA) and 1% penicillin-streptomycin. All these cells were maintained at 37° C in a humidified atmosphere containing 5% CO2 [66 (link)].

Skeletal muscle (C2C12) cells (Sigma Aldrich, Melbourne, Australia) were cultured in 10% fetal calf serum, and 1% Penicillin-Streptomycin-supplemented DMEM containing 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate (Sigma Aldrich, Melbourne, Australia) and grown in an optimal condition of 5% CO₂ at 37 °C. Once the cells reached 70% confluency, the C2C12 skeletal muscle myotubule phenotype was obtained by mitogen withdrawal (2% horse serum, Thermo Fisher, Melbourne, Australia) for 72 h. Three hours before the treatment, the C2C12 cells were exposed to a serum-free medium. Cells were then treated with various combinations of capsaicin, insulin, AICAR, and SB-452533 as per the following procedures.