Invadosome is required for myoblast fusion. (A and B) Differentiation and morphology of Tks5-depleted myoblasts. Tks5 was depleted by two lentiviral shRNAs and selected with puromycin for 3 d. After 3 d of DM treatment, cells were processed for immunoblotting (A) or immunostaining (B). Scale bar, 10 µm. (C and D) Myoblast fusion in Tks5-depleted myoblasts. Fusion efficiency was analyzed in day 4 DM-treated myoblasts by immunofluorescent staining and quantified as number of nuclei in multinuclear cell (nuclear number ≥3)/number of nuclei in MyHC-positive cell. Fusion efficiency is considered as 100% in control cells. Scale bar, 50 µm. All values reported in this study represent the mean ± SD of at least three independent experiments, and data were analyzed with one-way ANOVA. ***, P < 0.001.