Figure 7.

SIM+MET-induced necrosis in C4-2B metastatic CRPC cells is in part Ripk1 dependent. (a,b) Percentage cell viability (mean±S.D.) by the methylene blue assay in C4-2B3 and C4-2B4 cells following treatment with 50 μM Ripk1 inhibitor necrostatin-1 (Nec-1) or combination 4 μM simvastatin (SIM) and 2 mM metformin (MET)±25−100 μM Nec-1 for 72 h, n=3 separate experiments. *P<0.05, **P<0.01, ***P<0.001, NS=not significant, as determined by ANOVA followed by the Tukey multiple comparison procedure. (c) Treatment of C4-2B3 and C4-2B4 cells with 50 μM Nec-1 for 72 h reduces Ripk1–Ripk3 association. Immunoprecipitation of 600 μg protein from total cell lysates of C4-2B3 and C4-2B4 cells treated with 50 μM Nec-1 and/or combination 4 μM SIM and 2 mM MET was conducted with Ripk1 and Ripk3 antibodies followed by western blot with Ripk1, Ripk3, and GAPDH antibodies. No GAPDH detected in immunoprecipitates (not shown). Protein (40 μg) from total cell lysates were also immunoblotted as a control (input). Protein expression was quantified by densitometry normalized to GAPDH loading control (mean from two separate experiments). (d) Western blot analysis of HMGB-1 protein in 40 μl conditioned media from C4-2B3 and C4-2B4 cells following treatment with 25−50 μM Nec-1 or combination 4 μM SIM and 2 mM MET±25−50 μM Nec-1 for 48−72 h. HMGB-1 protein expression quantified by densitometry (mean from two separate experiments). ND, none detected. Ponceau S stain used to demonstrate equal loading