Figure 4. Notch2 knockdown confers protection against phenethyl isothiocyanate (PEITC)-induced apoptosis in LNCaP and PC-3 cells.

(A) Immunoblotting for cleaved Notch2 protein using lysates from LNCaP and PC-3 cells transiently transfected with a control (non-specific) siRNA or a Notch2-targeted siRNA and treated for 24 hours with dimethyl sulfoxide (DMSO) or 5 µM PEITC. Numbers above bands represent changes in protein levels relative to control siRNA-transfected cells treated with DMSO. Arrow (PC-3) identifies cleaved Notch2, the lower band is non-specific. (B) Histone-associated DNA fragment release into the cytosol (a measure of apoptosis) in LNCaP and PC-3 cells transiently transfected with a control siRNA or a Notch2-targeted siRNA and treated for 24 hours with DMSO (control) or 5 µM PEITC. Results are expressed as apoptosis enrichment relative to control siRNA-transfected cells treated with DMSO. (C) Immunoblotting for cleaved Notch1 and cleaved Notch2 proteins using lysates from LNCaP and PC-3 cells treated for 8 hours or 24 hours with DMSO (control) or 5 µM PEITC in the absence or presence of 50 µM DAPT. Numbers above bands represent changes in protein levels relative to corresponding DMSO-treated control. (D) Histone-associated apoptotic DNA fragment release into the cytosol in LNCaP and PC-3 cells treated for 24 hours with DMSO (control) or 5 µM PEITC in the absence or presence of 50 µM DAPT. Results are expressed as apoptosis enrichment relative to DMSO-treated control. Results (panels B and D) are mean ± SD (n = 3). *Significantly different (P<0.05) between the indicated groups by one-way ANOVA followed by Bonferroni's multiple comparison test. Each experiment was repeated twice, and representative data from one such experiment are shown.