PUMA Regulates BITC-Induced Apoptosis Figure 5. Opposing effect of BITC treatment on levels of Bcl-xL and Bcl-2 proteins in MDA-MB-231 cells. (A) Western blotting for Bcl-xL and Bcl-2 using lysates from MDA-MB-231 cells treated with DMSO (control) or BITC (2.5 or 5 mM) for the indicated time periods. Number above band indicates change in level compared to the corresponding DMSO-treated control. (B) Western blotting for Bcl-xL and (C) quantitation of histone- associated DNA fragment release into the cytosol in MDA-MB-231 cells transiently transfected with empty vector or vector encoding for Bcl-xL and treated for 24 h with DMSO or 5 mM BITC. Results are expressed as enrichment relative to corresponding DMSO-treated control. Data represent mean 6 SD (n = 3). Significantly different (P,0.05) acompared with corresponding DMSO-treated control, and bbetween empty vector transfected cells and Bcl-xL overexpressing cells by one-way ANOVA followed by Bonferroni’s multiple comparison test. (D) Western blotting for Bcl-2 and (E) quantitation of histone-associated DNA fragment release into the cytosol in MDA-MB-231 cells transiently transfected with a control siRNA or the Bcl-2-targeted siRNA and treated for 24 h with DMSO or 5 mM BITC. In panel E, results shown are relative to control siRNA transfected cells treated with DMSO. Data represent mean 6 SD (n = 3). Significantly different (P,0.05) acompared with corresponding DMSO-treated control, and bbetween control siRNA transfected cells and Bcl-2 siRNA transfected cells by one-way ANOVA followed by Bonferroni’s multiple comparison test. (F) Western blotting for p53 and Bcl-2 using lysates from MCF-7 cells transiently transfected with a control siRNA or the p53-targeted siRNA and treated for 24 h with DMSO or 5 mM BITC. (G) Western blotting for p53 and PUMA using lysates from MCF-7 cells transiently transfected with a control siRNA or the p53-targeted siRNA and treated for 24 h with DMSO or 5 mM BITC. Number above the bands in 5F, G represents change in level relative to control siRNA transfected cells treated with DMSO. All the experiments were repeated at least twice and representative data from one such experiment are shown. doi:10.1371/journal.pone.0032267.g005 PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e32267