RNAi-mediated depletion of Bnip-2 reduces myogenic differentiation. (A) Lysates of C2C12 cells stably transfected with pSilencer containing one of three independent Bnip-2 siRNA sequences (designated 1, 3, and 4) or pSilencer containing an irrelevant sequence (−) were Western blotted with Bnip-2 or, as a control, pan-cadherin antibodies. (B) Photomicrographs of C2C12 cells that express Bnip-2 siRNA sequences or an irrelevant sequence (pSilencer) as indicated, cultured in DM, fixed, and stained with an antibody to MHC. Bar, 0.5 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results. Asterisks indicate difference from pSilencer control, P < 0.01. A level of myotube formation by control cells (∼80% nuclei in MHC⁺ cells) was selected so as to permit visualization of diminished differentiation by Bnip-2 siRNA. (D) Western blot analysis of C2C12 cells that express Bnip-2 siRNA sequences (+) or an irrelevant sequence (−) cultured in GM (G) or in DM for the indicated times.