Fig. 5: OSW-1-induced necroptosis is executed by CaMKIIδ-mediated MLKL phosphorylation.

A Western blotting of indicated proteins in HCT116 cells treated with OSW-1 (0.5 nM) at indicated time points. B Western blotting of indicated proteins in HCT116 cells treated with OSW-1 at indicated concentrations for 24 h. C, D Western blotting of indicated proteins in (C) WT and p53-KO, D WT and PUMA-KO HCT116 cells treated with OSW-1 (0.5 nM) for 24 h. E MTS analysis of HCT116 cells treated for 48 h with OSW-1 at indicated concentrations alone or in combination with the CaMKII inhibitor KN93 (1 μM). F–I HCT116 cells were treated for 24 h with OSW-1 (0.5 nM) alone or in combination with KN93 (1 μM). F Crystal violet staining of viable cells. G ATP levels in treated cells. H LDH release from treated cells. I Western blotting of indicated proteins in whole cell lysates (WCL) and HMGB1 in 20-μl cell culture medium (M). J, K WT and CaMKIIδ-KO J HCT116 and K RKO cells were treated with OSW-1 as in (C). Necroptosis was analyzed by measuring ATP levels (upper), Western blotting of indicated proteins (middle), and crystal violet staining (lower). L HCT116 cells treated with OSW-1 as in (C) were subjected to IP to pull down CaMKII (left) or MLKL (right), followed by western blotting of indicated proteins. IgG was used as a negative control. Quantitative results in (E, G, H, J, K) were expressed as means ± s.d. of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.