Figure 4

p53-independent PUMA upregulation by PAC-1 promotes mitochondria-mediated apoptosis. (a) Western blot analysis indicated that p53 was upregulated in multiple cancer cells upon PAC-1 treatment. (b) PAC-1 induced significant nuclear condensation in HCT116 and p53 KO HCT116 cells as seen in representative microscopic images and graph (n=3, mean±S.D.). Western blot established the knockout status of p53 in HCT116. (c) Clonogenic cell survival assay revealed that PAC-1 effectively killed HCT116 p53 KO cells, although slightly lesser than its WT counterpart. Representative images and graph are given (n=3, mean ±S.D.). (d) PAC-1 induced upregulation of PUMA in multiple cancer cell lines. As seen in blot, PUMA is upregulated even in the absence of p53 (HCT116 p53 KO). Bim and Bik were also upregulated in SiHa cells but not in MCF7 and HCT116 cells, suggesting cell specificity of the response. (e) A representative western blot indicating silencing of PUMA in MCF7 cyt. c-EGFP cells. (f) Cell death was analyzed by PI staining using FACS in MCF7 cyt. c-EGFP cells as well as PUMA-silenced MCF7 cyt. c-EGFP cells. As noticed, silencing of PUMA inhibited PAC-1-induced cell death significantly. The percentages of PI-positive cells are mentioned. (g) Silencing of PUMA inhibited cyt. c release from the mitochondria and nuclear condensation in MCF7 cyt. c-EGFP cells upon PAC-1 treatment. The representative fluorescent microscopic images are shown (scale bar: 50 μm). The diffuse pattern of EGFP in cells represents release of cyt. c from the mitochondria