Fig. 4. shRNA against caspase-9 impairs muscle differentiation. C2C12 cells were transduced with either an adenoviral vector encoding shRNA targeted against caspase-9 (AdshRNA-C9) or the same vector without the shRNA (AdNull). Caspase-9 levels were assessed by immunoblot (A) and the effect of shRNA on caspase-3-like activity was assessed by DEVD-afc (B). Enzyme activity is expressed as an increase in arbitrary fluorescent units per minute per milligram of protein (AFU/min/mg). (C) The effect of AdshRNA-C9 knockdown on caspase processing was determined by immunoblot. Arrows indicate the processed forms of caspase-9 and caspase-3. (D) The effect of AdshRNA-C9 knockdown on cell fusion (see Materials and Methods) was assessed. The histograms show the mean ± s.d. of three separate experiments. (E) Top, western blot for caspase-9 in stained samples. Bottom, MyHC (green) staining with DNA (blue) counterstaining (Hoechst 33342).