PUMA Regulates BITC-Induced Apoptosis Figure 6. BITC administration increase PUMA expression in vivo in MDA-MB-231 xenografts. Immunohistochemical analysis for (A) PUMA and (C) Bcl-xL in MDA-MB-231 tumor sections from control and BITC-treated mice (7). Magnification- 2006; scale bar- 40 mm. Immunoblotting for (B) PUMA and (D) Bcl-xL using tumor supernatants from control and BITC treatment groups. Lower panels in B and D represent densitometric quantitation (arbitrary units). Statistical significance was determined by Student’s t-test (n = 5 for control and n = 3 for BITC treatment group). doi:10.1371/journal.pone.0032267.g006 sensitive to BITC-mediated induction of PUMA protein compared with the MDA-MB-231 cell line. Further studies are needed to gain insights into the p53-independent mechanism(s) responsible for BITC-mediated induction of PUMA protein especially in the MDA-MB-231 cells. It is possible that this effect in MDA-MB-231 cells is mediated by transcriptional suppression of Slug, which is a negative regulator of PUMA [31]. We have shown previously that BITC-mediated inhibition of epithelial-mesenchymal transi- tion in MDA-MB-231 cells is associated with transcriptional repression of Slug [15]. At the same time, several other transcription factors have been implicated in regulation of PUMA expression, including p53 homologue p73 [32], the forkhead family member FOXO3a [33], c-Myc [34], and E2F1 [35], and their involvement in BITC-mediated induction of PUMA protein can’t be excluded. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells is accompanied by an increase in G2/M phase cell cycle arrest, which is accompanied by induction of p21. These findings are consistent with literature data showing the balance between induction of PUMA and/or suppression of p21 in cells committed for cell cycle arrest or apoptotic death [36]. Conversely, suppression of PUMA [31] or selective induction of PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e32267