FIG 5.

Fst rescued defective skeletal myogenesis in Lgr4-KD C2C12 cells. (A) Fst expression in undifferentiated Lgr4-KD C2C12 cells treated with control BSA (200 ng/ml) or RSPO2 (200 ng/ml) for 1 day was determined by qRT-PCR. Fst expression was normalized to Gapdh expression. Samples were prepared in triplicate. (B to D) Exogenous FST protein rescued myogenic defects in Lgr4-KD C2C12 cells. Immunofluorescence staining for MyHC expression in Lgr4-KD C2C12 cells and control eGFP-shRNA cells cultured in the presence of control BSA (500 ng/ml) and FST (500 ng/ml). Cells were harvested at differentiation day 5. (E) The percentages of nuclei in MyHC-positive cells relative to the total number of nuclei and percentages of MyHC-positive cells containing a single nucleus, 2 to 5 nuclei, 6 to 10 nuclei, and more than 10 nuclei were calculated. Experiments were performed in duplicate, and more than 1,000 total nuclei were counted. Error bars indicate SEM. P values were calculated by Student's t test. **, P < 0.01; *, P < 0.05.