Fig. 4
Effect of HX on proliferation capacity and myogenic commitment of C2C12. Mouse myoblasts C2C12 were seeded 1 × 104 cells/well in GM for 1, 4 and 7 days under both NX and HX conditions. a MTT assay shows cell viability up to day 7 under HX compared to NX condition. The absorbance was examined at wavelength 570 nm. b SRB assay indicative for total protein contents reveals the cell number after days 1, 4 and 7 under HX compared to NX condition. c Measurement of C2C12 colony size (µm2) at 1 × 102, 5 × 102 and 1 × 103 seeding densities under HX compared to NX condition. C2C12 were seeded at various densities in GM in T-25 culture flasks (n = 3 per experimental group) for 7 days under both NX and HX conditions. The colonies were fixed in 4% PFA for 10 min and then were stained with 5 mg/mL crystal violet in 2% ethanol for 8 min. d–i Evaluation of the myogenic commitment of C2C12 under HX. C2C12 were seeded 1 × 104 cells/well in triplicates with myogenic differentiation medium up to day 7 under both NX and HX conditions. d Number of MyoD positive cells after day-1 and day-4 post-induction. e Number of Myogenin positive cells at day-7 post-induction. f–i Representative immunofluorescence shows MyoD (red) and Myogenin (red) positive cells (arrow) after 3 days under both NX and HX conditions. All data presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0. 001. DAPI was used as a nuclear counterstain (blue). Scale bar = 20 µM