Fig. 3: PLEKHO2 deficiency facilitates necroptosis and activation of RIPK1.

A Control and PLEKHO2 reexpressed PLKEHO2−/− MEFs were treated with DMSO, TCZ, TCZN, TSZ, and TSZN for 8 h, cell viability was measured by ATP level. Abbreviations are as follows: TSZ TNF-α (20 ng/ml) + Smac mimetic (100 nM) + zVAD (20 μM), TSZN TNF-α + Smac mimetic + zVAD + Necrostatin-1 (20 μM), TCZ TNF-α (20 ng/ml) + CHX (10 μg/mL) + zVAD (20 μM), TCZN TNF-α + CHX + zVAD + Necrostatin-1 (20 μM). B Control and PLEKHO2 reexpressed PLKEHO2−/− MEFs treated with DMSO, TS, TSN, TC, and TCN for 8 h, cell viability was measured by ATP level. Abbreviations are as follows: TS TNF-α (20 ng/ml) + Smac mimetic (100 nM), TSN TNF-α + Smac mimetic + Necrostatin-1 (20 μM), TC TNF-α + CHX (20 ng/ml), TCN TNF-α + CHX + Necrostatin-1 (20 μM). C WT and PLKEHO2−/− MEFs were treated with TCZ, TCZ, TNF-α (20 ng/ml) + CHX (10 μg/mL) + zVAD (20 μM) for indicated periods of time. Cell lysates were probed with indicated antibodies. D WT and PLKEHO2−/− MEFs treated with TSZ, TNF-α (20 ng/ml) + Smac mimetic (100 nM) + zVAD (20 μM) for indicated periods of time. Cell lysates were probed with indicated antibodies. E WT and PLKEHO2−/− MEFs were treated with Flag-TNF-α (100 ng/ml) for the indicated periods of time, complex I was immunoprecipitated using anti-FLAG beads, endogenous RIPK1 ubiquitination, and indicated proteins were detected by western blotting. F Control and PLEKHO2 reexpressed PLKEHO2−/− MEFs were treated with TCZ, TCZ, TNF-α (20 ng/ml) + CHX (10 μg/mL) + zVAD (20 μM) for 3 h, and then Caspase-8 was immunoprecipitated (IP). IP and cell lysates were analyzed by western blotting using the indicated antibodies. Results are representative of at least three independent experiments.