Murine Muscle Progenitor Cells Culturing and Stimulation

According to the previous description, murine MPCs were collected from the hind limb muscle of neonatal B6 mice (Sciorati et al., 2015 (link)). MPCs were cultured in Dulbecco’s modified Eagle’s medium Nutrient/F12 (DMEM/F12, Hyclone, United States) containing with 10% fetal bovine serum (FBS, Gibco, United States), 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate in a 5% CO₂-humidified chamber (Heraeus, Germany) at 37°C. For differentiation studies, MPCs were cultured in differential medium (DM) (DMEM, added with 2% horse serum) for 72 h. For pro-inflammatory stimuli, MPCs were treated with IFN-γ (60 ng/mL, R&D, United States) for 24 h (Nozaki et al., 2010 (link)). For inhibiting UPR and the branches, the cells were co-cultured with 4-PBA (10 mM, Selleck, China), 4μ8c (50 μM, Selleck, China), and GSK2606414 (1 mM, Selleck, China), respectively, under pro-inflammatory stimuli. TM (1 μg/mL, Santa Cruz, United States), or TG (0.2 mmol/L, Santa Cruz, United States) were used for 4 h as positive control for testing UPR activation and as UPR activators. Cells were cultured in DM with or without 20 mM SB202190 (Sigma, United States) to blocking p38 activation in IFN-γ medium.