Fig. 3.

CMKLR1 knockdown impairs fusion of C₂C₁₂ myoblasts into myotubes. An anti-myosin heavy chain (MHC) antibody (red) was used to detect MHC protein in undifferentiated C₂C₁₂ myoblasts (A) and in differentiating C₂C₁₂ cells at the indicated differentiation time points following transduction with 500 multiplicity of infection of CMKRL1 shRNA (CR500)- and LZ-shRNA (LZ500) or vehicle (VEH) treatment (B). The cell nuclei were counterstained with Hoechst 33258 (blue). Each image is representative of 4 independent samples; scale bar = 200 μm. On day 8, F-actin filaments were stained with phalloidin (red) to visualize the formation of myotubes, and the cells were counterstained with Hoechst 33258 nuclear stain (blue) (C). Each image is representative of a total of 6 samples obtained from 2 independent experimental replicates; scale bar = 200 μm. The myogenic index was calculated as the ratio of nuclei in MHC-positive myotubes/total nuclei in the field of view (D). P < 0.05 compared with the VEH and LZ-shRNA controls at the indicated time point (*) and compared with the respective LZ-shRNA control only (†), two-way ANOVA followed by Bonferroni multiple-comparison test.