Figure 6.

Nesprin-1 mutants cause defects in myoblast differentiation. IF showed that exogenously expressed V5-tagged WT-1α₂ or mutants and dominant genative-1KASH were localized at the NE. GFP was expressed as a reporter for retroviral infection (A). Upon differentiation, fewer multinucleated myotubes were observed in cells transduced with mutant R8272Q and dominant negative-1KASH compared with C2C12 cells transduced with eGFP alone (MIG only) or nesprin-1α₂ WT (B). The fusion index was reduced in cells transduced with the mutant R8272Q and dominant negative-1KASH compared with 1α₂ WT (C), more than 600 nuclei for each clone were counted by microscopy (63× objective) at day 6, three independent experiments were performed for each clone. Further analysis of the MHC positive multinucleated populations revealed that myotubes expressing nesprin-1 mutants contained fewer nuclei compared to the controls (D). qPCR and WB showed that nesprin-1 mutant R8272Q and dominant negative-1KASH caused significant reduction of myogenin (E, F) and MHC (E, G) levels at DM day 2 and day 6 respectively, qPCR also showed that dominant negative-1KASH caused significant reduction of MyoD (E) levels at DM day 2. All were normalized to GFP, three independent experiments were performed shown as mean ± SEM, *P < 0.05 using Student’s t-tests or two-way ANOVA analysis.