Figure 2.

GSH depletion impairs differentiation of C2C12 cells. Myoblasts were grown in growth medium to reach confluency and 24 hours later the culture medium was switched to differentiation medium to induce myogenesis. Twenty-four hours after myogenic stimulation in differentiation medium cells were treated with DEM (0.8 mmol/L preincubation for 15 minutes) followed by BSO (1 mmol/L) for 24 hours to deplete GSH and inhibit γ-GCS activity (DEM+BSO, open bars) or not (control, closed bars), after which cells were grown in differentiation medium until day 7. A: Total GSH levels were assessed by HPLC as in Figure 1B. B: To assess the effect of DEM/BSO on differentiation and myogenesis, CK activity was determined in cell lysates at different times of culture in differentiation medium with or without DEM+BSO preincubation. C: The morphological appearance of myotubes at day 7 was examined by confocal microscopy. Nuclei were stained by propidium iodide (red) and MyHC by an anti-MyHC antibody followed by a FITC-labeled secondary antibody (green). In some cases we examined the effect of GSH replenishment by GSHEE on DEM+BSO-treated cells. D: Quantitation of myotube formation from the representative images shown in C as the percentage of nuclei in MyHC-positive cells compared to total number of nuclei in the field. E: Cell survival was determined after treatment with DEM+BSO as the percentage of LDH released into the medium and viability expressed as percentage of LDH found in cells plus medium. Data are the mean ± SD of three to five independent experiments. *, P < 0.05 versus untreated control cells cultured in DM medium.