Altered expression of differentiation markers and kindlin-2 knockdown. A. Relative RNA levels of myh8 and myotilin in RNAi transfected, 4 day differentiated C2C12 cells as determined by qPCR. Each value was calculated from 3 independent transfections with qPCR performed in triplicate. In kindlin-2 RNAi transfected cells, myh8 and myotilin levels were 58 +/-1% and 56+/-3%, respectively, of the values obtained for control cells. B. Immunoblot on protein extracted from day 4 differentiated samples transfected with scrambled or kindlin-2 RNAi. Blots were probed with the following antibodies: myogenin (myog), myosin heavy chain (MHC), and GAPDH (loading control). Densitometric analysis revealed no difference in myogenin levels between samples, and a reduction to 75% of normal for MHC. C. Brightfield images of scrambled (scr) or kindlin-2 (kind2) RNAi transfected C2C12 cells incubated in differentiation media for 6 days (DM D6). Note extensive myotube formation in the scrambled sample as compared to the kindlin-2 RNAi sample. D. Immunoblot of day 4 (DM D4) and day 6 (DM D6) differentiated myocytes transfected with scrambled (scr) or kindlin-2 (kind2) RNAi. Blots were probed with antibodies to β1D integrin and GAPDH. Densitometric analysis revealed 75% and 77% reductions respectively in β1D integrin levels in kindlin-2 RNAi samples.