PUMA Regulates BITC-Induced Apoptosis Figure 1. BITC treatment increases PUMA protein level in MCF-7 and MDA-MB-231 cells. (A) Western blotting for PUMA using lysates from MCF-7 and 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 corresponding DMSO-treated control. (B) Immunofluorescence microscopy for PUMA in Mito GFP expressing MCF-7 cells after 24 h treatment with DMSO or 5 mM BITC (1006 objective magnification). Arrow represents localization of PUMA in mitochondrion. (C) Immunoblotting for PUMA using lysates from MCF-7 cells transiently transfected with a control siRNA or PUMA-targeted siRNA and treated for 24 h with DMSO or 5 mM BITC. (D) Quantitation of histone-associated DNA fragment release into the cytosol (a measure of apoptosis) in MCF-7 cells transiently transfected with a control siRNA or PUMA-targeted siRNA 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 = 2–3). Significantly different (P,0.05) compared with arespective DMSO-treated control and bbetween control siRNA transfected and PUMA siRNA transfected cells by one-way ANOVA followed by Bonferroni’s multiple comparison test. The experiments were repeated twice and data from one representative experiment are shown. doi:10.1371/journal.pone.0032267.g001 Bcl-xL and Bcl-2 protein levels in MCF-7 cells. Ectopic expression of Bcl-xL through transient transfection in MCF-7 cells (Fig. 4B) conferred partial but statistically significant protection against BITC-induced apoptosis (Fig. 4C). Similarly, Bcl-2 overexpressing MCF-7 cells (Fig. 4D) were significantly more resistant to BITC- mediated enrichment of histone-associated DNA fragment release into the cytosol compared with cells transiently transfected with the empty vector (Fig. 4E). These results indicated that BITC- induced apoptosis in MCF-7 cells was caused by downregulation of both Bcl-xL and Bcl-2 proteins. Opposing effect of BITC on Bcl-xL and Bcl-2 protein levels in MDA-MB-231 cells Similar to MCF-7 cells (Fig. 4A), BITC treatment caused a decrease in protein level of Bcl-xL in MDA-MB-231 cells especially at the 5 mM concentration (Fig. 5A). To the contrary, BITC-treated MDA-MB-231 cells exhibited a marked increase in level of Bcl-2 protein (Fig. 5A). The BITC treatment downreg- ulated Bcl-xL protein expression in MDA-MB-231 cells transiently transfected with both the empty vector and vector encoding for Bcl-xL (Fig. 5B). In agreement with results in MCF-7 cells PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e32267