Figure 6.

siRNA knockdown of BAD expression abolishes rotenone-induced apoptosis in SH-SY5Y cells. A, Transfection of siBAD, but not siNS, reduces BAD protein expression. SH-SY5Y cells were transfected with a 100 nm concentration of an siRNA specific to human BAD or a FITC-labeled doubled-stranded RNA oligomer control. BAD protein expression was analyzed 2 d later by Western blotting. The relative expression levels of BAD were normalized to the loading control β-actin. B, Reduced BAD expression renders SH-SY5Y cells resistant to rotenone. Cells were transfected with siRNA as in A. Cells were also cotransfected with plasmid DNA encoding eGFP to label transfected cells. Two days after transfection, cells were pretreated with 10 ng/ml bFGF or vehicle control for 1 h, followed by 50 nm rotenone or vehicle control for 24 h. Apoptosis were scored in eGFP-positive cell population. Data are representative of three independent experiments. At least 600 transfected cells were counted in each data point. Error bars are SEM. ***p < 0.001; ns, not significant (ANOVA).