Fig. 2: OSW-1-induced necroptosis requires p53-mediated transcriptional upregulation of PUMA.

A Comparison of OSW-1 IC₅₀ of p53-wildtype (WT) and p53-mutant CRC cell lines analyzed in Fig. 1A. B, C HCT116 cells were treated with (B) OSW-1 (0.5 nM) for indicated time, or (C) OSW-1 at indicated concentrations for 24 h. Upper, real time RT-PCR analysis of PUMA mRNA expression; lower, western blotting of indicated proteins in whole cell lysates (WCL) and HMGB1 in 20-μl cell culture medium (M). D, E Western blotting of indicated proteins in D WT and p53-knockout (p53-KO), E WT and PUMA-KO HCT116 cells treated with OSW-1 (0.5 nM) for 24 h. F MTS analysis of WT, p53-KO, and PUMA-KO HCT116 cells treated with OSW-1 at indicated concentrations for 48 h. G-I WT, p53-KO, and PUMA-KO HCT116 cells were treated with OSW-1 as in (D) and analyzed by (G) crystal violet staining of viable cells, H annexin-V/PI staining followed by flow cytometry, I measuring ATP levels, and J TEM. In (J), representative TEM images are shown. White arrowheads indicate plasma membrane, and black arrowheads denote mitochondria. Scale bars, 2 μm. K, L WT and PUMA-KO HCT116 cells with or without infection with an adenovirus expressing PUMA (Ad-PUMA) or control BH3-deleted PUMA (Ad-control) were treated with OSW-1 as in (D). K Crystal violet staining of viable cells. L Western blotting of indicated proteins. Quantitative results in (B, C, F, I) were expressed as means ± s.d. of two or three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.