Fig. 1. The inhibitory properties of primidone in the course of RIPK1-mediated necroptosis are species-independent.

A Human U937 cells were stimulated at 37 °C for up to 8 h with 100 ng/ml TNFα + 1 μM SMAC mimetic SM-164 + 25 μM zVAD (TSZ) in the absence or presence of 1 mM primidone, wherein the drug was added 30 min before the induction of necroptosis. B U937 cells were treated for 8 h with 100 ng/ml TNFα + 1 μM SMAC mimetic SM-164 + 25 μM zVAD (TSZ) in the absence or presence of primidone (concentrations indicated), wherein the drug was added 30 min before the induction of necroptosis. Necroptotic cell death was quantified by FACS analysis using 7-AAD and phosphatidylserine accessibility (Annexin V staining) as markers. Each graph shows the mean ± SD; n = 3 independent repeats. Each sample analyzed in the time course of this experiment was split for the corresponding western blotting [illustrated in (C)]. C The expression levels and activation status (phosphorylation) of the indicated necrosome member RIPK1, RIPK3, and MLKL after the induction of necroptosis in the absence or presence of primidone were analyzed by western blotting, using the indicated specific antibodies. The blots were stripped and re-probed with an antibody against HSP90 as the loading control. For each cell line illustrated herein, one representative cropped blot of three independent experiments is shown. Human HT-29 cells (D) and primary MEFs (G) were treated as described under (A) and the activation status of the indicated necrosome members was analyzed as described under (C). Murine NIH3T3 (E) and L929 cells (F) were stimulated at 37 °C for up to 8 h with 100 ng/ml TNFα + 25 μM zVAD (TZ) in the absence or presence of 1 mM primidone, wherein primidone was added 30 min before the induction of necroptosis. Cell death was quantified and detection of the activation status (phosphorylation) of the indicated necrosome members was detected as described before.