Fig. 5. Transfection of C2C12 myoblasts with mutants of lamin A prevents nuclear envelope organization and inhibits myoblast differentiation. C2C12 cells grown on glass coverslips were transiently transfected with wild-type lamin A or lamin A with point mutation W520S. 24 hours after transfection, cells were either incubated in growth medium for next 24 hours (undifferentiated, a) or induced to differentiate with 2% horse serum (differentiated, b). The cultures were fixed and co-stained for the presence of the exogenous lamin A (with anti-FLAG antibodies) and endogenous myogenin (a,b), endogenous lamin B1 (c,d) or endogenous lamin C (e,f). (a,b) Examples of nuclei scored as having nucleoplasmic staining (see Fig. 6) are indicated with ^* or +; examples of cells scored as having nuclear envelope staining are indicated with @. Following transfection with any FLAG vector, differentiation events are delayed by approximately 48 hours. However, cultures transfected with FLAG-W520S did not differentiate at all. Images were obtained using a BioRad laser sharp 2000 confocal microscope. Bars, 10 µm.