FIG 4.

Depletion of Fst compromised RSPO2-enhanced skeletal myogenesis in C2C12 cells. (A) Fst expression in undifferentiated (U) and differentiating (D1 and D3) C2C12 cells treated with control BSA (200 ng/ml) or RSPO2 (200 ng/ml) for 1 and 3 days was determined by qRT-PCR. Fst expression was normalized to Gapdh expression. Samples were prepared in triplicate. (B) Depletion of Fst expression by shRNA in C2C12 cells determined by qRT-PCR. Gapdh expression was used for normalization. Samples were tested in duplicate. (C to E) Immunofluorescence staining for MyHC expression in Fst-KD C2C12 cells and control eGFP-shRNA cells cultured in the presence of control BSA (200 ng/ml) and RSPO2 (200 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 < 0.001; **, P < 0.01.