α-Actin overexpression in MBs lacking Srf restores heterotypic fusion. (A) Phase-contrast representative pictures of FACS-sorted control and Mut/Act⁺ SCs cultured in rich medium (D0) or 3 d after differentiation induction (D3). (B) Proportion of nuclei within multinucleated cells (fusion index) in control, mutant, and Mut/Act⁺ cells 3 d after differentiation (n = 4–7). (C) Mean number of nuclei per MyHC⁺ cell in control, mutant, and Mut/Act⁺ cells induced to differentiate for 3 d (n = 4–5). (D) MB control, MB mutant, or MB Mut/Act⁺ were labeled with Green Cell Tracker and mixed with control MBs labeled with Orange Cell Tracker. After 48 h of co-culture in differentiation medium, the percentage of dual-labeled MTs per total number of nuclei was scored (n = 4–7). (E) MT control were labeled with Cell Tracker 1 (Orange; visualized in red) and mixed with MB control, MB mutant, or MB Mut/Act⁺ labeled with CellTracker 2 (Deep Red; visualized in green). After 48 h of co-culture, MTs were analyzed for dual labeling (indicated by arrow). (F) MT control were labeled with Orange Cell Tracker and mixed with MB control, MB mutant, or MB Mut/Act⁺ labeled with Deep Red Cell Tracker. The percentage of dual-labeled cells per total number of cells was scored (n = 4–5). Data are mean ± SEM. **, P < 0.01.