Serum fbs

Freshly isolated satellite cells were seeded at 3,000 cells/cm² in growth medium containing 1:1 DMEM:MCDB (Gibco and Sigma-Aldrich, respectively) supplemented with 20% serum FBS (Gibco) and 1% Ultroser G (Pall) on Matrigel coated flasks (BD Biosciences) and cultured in an incubator under physiological oxygen pressure (37 °C, 6.5% CO₂, 3% O₂). To assess proliferation, cells were pulsed with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU, 1 × 10⁻⁶ M), 2 h prior to fixation (ThermoFisher Click-iT Plus EdU kit, C10640). For differentiation experiments, 60 h after plating, medium was replaced to remove Ultroser G, and cells were cultured for a total of 7 days to reach early differentiation.

Isoalantolactone (IAL) was obtained from Chengdu PuFeiDe Biotech Co., Ltd. (Chengdu, China). 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO) and N-methyl-4-phenylpyridinium iodide (MPP⁺ iodide) were purchased from Sigma Aldrich (St Louis, MO, USA). RA (nrf2 inhibitor) and MK2206 (AKT inhibitor) were obtained from Selleck Chemicals Aldrich (Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM), trypsin (0.05% and 0.25%) and serum (FBS) were obtained from Gibco (Grand Island, NY, USA).