FIG 2.

Depletion of Lgr4 expression attenuated RSPO2-enhanced myogenic differentiation and WNT/β-catenin signaling. (A) Immunofluorescence staining for the MyHC protein in differentiating control and Lgr4 KD C2C12 cells treated with RSPO2 (200 ng/ml) for 4 days. (B) The myogenic differentiation index was calculated by dividing the number of nuclei in MyHC-positive cells by the total number of nuclei and expressing the result as a percentage. (C) The cell fusion index was presented as a distribution of MyHC-positive cells based on their nucleus number. (D) Activation of WNT/β-catenin signaling was measured by the activity of the super TopFlash reporter. Control and Lgr4-KD C2C12 cells in 24-well plates were transfected with the TopFlash reporter (350 ng/well) and control Renilla luciferase reporter (150 ng/well) plasmid DNAs and stimulated with the RSPO2 (25 ng/ml) and WNT3A (50 ng/ml) proteins for 24 h. Luciferase activity was measured and normalized to the activity of control Renilla luciferase. Samples were prepared in triplicate. Error bars indicate SEM. P values were calculated by Student's t test. ***, P < 0.001; **, P < 0.01.