Fig 2: Caspase-1 drives Bid processing during GSDMD-independent secondary necrosis.(A) Immunoblots showing caspase-1, caspase-7, caspase-3, caspase-8, and caspase-9 processing in WT, Asc-/-, Casp1-/-/Casp11-/-, and Gsdmd-/- primary BMDMs after transfection with poly(dA:dT). (B) LDH release from WT, Asc-/-, Casp1-/-/Casp11-/-, Gsdmd-/-, and Gsdmd-/-/Casp8-/- iBMDMs after transfection with poly(dA:dT). (C) Immunoblots showing caspase-3 processing in Gsdmd-/- and Gsdmd-/-/Casp8-/- iBMDMs after transfection with poly(dA:dT). (D) Immunoblots showing Bid cleavage in Gsdmd-/- and Gsdmd-/-/Casp8-/- iBMDMs after transfection with poly(dA:dT). (E) Immunoblots showing Bid cleavage in Gsdmd-/- and Gsdmd-/-/Casp1-/- iBMDMs after transfection with poly(dA:dT). (F) In vitro cleavage assay showing processing of recombinant Bid by recombinant caspase-1. Graphs show mean ± SD. * “ns,” no significance (unpaired t test). Data and blot are representative of at least three independent experiments.

Fig 3: The CASP8(?E385) promotes necroptosis in vitro.A Primary WT and Casp8?E385/?E385 MDFs were treated with TNF-a (20 ng/ml)+Smac (1 µM)+zVAD (20 µM) (TSZ) and TNF-a + Smac + zVAD + Nec-1 (30 µM) (TSZN) for 6.45 h, TNF-a + CHX (20 µg/ml)+zVAD (20 µM) (TCZ) and TNF-a + CHX + zVAD + Nec-1 (30 µM) (TCZN) for 4.45 h. Bars, mean ± SD. P values above the asterisk (unpaired, two-tailed t test) ****p < 0.0001. B Primary WT and Casp8?E385/?E385 bone marrow derived macrophages (BMDMs) were treated with LPS (100 ng/ml), LPS + zVAD (20 µM) (LZ), LPS + zVAD + Nec-1 (30 µM) (LZN), poly(I:C) (100 µg/ml), poly(I:C) + zVAD (20 µM) (PZ), poly(I:C) + zVAD + Nec-1 (30 µM) (PZN), TNF-a + Smac + zVAD (TSZ), TNF-a + Smac + zVAD + Nec-1 (TSZN) for 3 h. Bars, mean ± SD. P values above the asterisk (unpaired, two-tailed t test) ***p < 0.001, ****p < 0.0001. C Immunoblotting of the indicated protein expression in primary WT and Casp8?E385/?E385 MDFs which were treated with TNF-a (20 ng/ml) +Smac (1 µM) +zVAD (20 µM) (TSZ) for the indicated time. D Immunoblotting of primary WT and Casp8?E385/?E385 MDFs which were treated with TNF-a (20 ng/ml) +Smac (1 µM) +zVAD (20 µM) (TSZ) for the indicated time. E WT and Casp8?E385/?E385 MDFs were treated with TNF-a (40 ng/ml)+CHX (40 µg/ml)+zVAD (50 µM) for the indicated time, complex II was immunoprecipitated using anti-RIPK1, the recruitment of RIPK3, FADD and caspase-8 were detected by western blotting. F Primary WT and Casp8?E385/?E385 BMDMs were treated with LPS (200 ng/ml)+zVAD (40 µM) followed by western blot and immunoprecipitation. G Primary WT and Casp8?E385/?E385 MDFs were treated with TNF-a (40 ng/ml)+CHX (40 µg/ml) followed by western blot and immunoprecipitation.

Fig 4: The addition of SM to TNF/Mel sensitises cells to RIPK1-dependent cell death ACell viability analysis using CellTiter-Glo of BN175 cells treated with the indicated agents for 24 and 48 h (n = 4 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). SM represents SM-164. Error bars represent SD and statistical analysis was performed with an unpaired t-test, ****P = 0.0001. Exact P values are shown in Appendix Table S1.BTNF-induced complex-II immunoprecipitation. BN175 cells were treated with the indicated agents for 8 h. FADD immunoprecipitation was performed followed by Western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).CProximity ligation assay between Ripk1 and Casp-8 performed in BN175 cells upon treatment with the indicated agents for 8 h (n = 3 biological replicates, shown are representative images). Scale bar represents 10 µm. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).DDEVDase activity assay of BN175 cells treated with the indicated drugs for 6 h (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). Error bars represent SD, and statistical analysis was performed with an unpaired t-test, ****P = 0.0001. Exact P values are shown in Appendix Table S1.ECell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 knockouts (KO) cells treated with the indicated agents for 18 and 48 h (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. SM represents SM-164. Error bars represent SD, and a one-way ANOVA was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), ****P = 0.0001. Exact P values are shown in Appendix Table S1. Source data are available online for this figure.

Fig 5: The embryonic lethality caused by D325A mutation in Ripk1 occurs earlier than that triggered by Casp8 knockout in mice.(A) Genetic analysis of offspring from intercrosses of Casp8+/− and Ripk1D325A/+ parents, respectively. *: less and thinner YS vessels and signs of hyperemia in the abdominal area in E11.5 embryos; **: severe YS vascularization defect and dead EP in E12.5 embryos; #: defective vascularization in E10.5 YS and normal EP; ##: no vessels in E11.5 YS and severe abdominal hemorrhage in the EP; ###: dead and resorbed embryos at E12.5. (B) Representative images of E10.5, E11.5, and E12.5 embryos described in (A). Scale bars, 1 mm. See also S1 Fig. E10.5, embryonic day 10.5; E11.5, embryonic day 11.5; E12.5, embryonic day 12.5; EP, embryo proper; Ripk1, receptor interacting serine/threonine kinase 1; YS, yolk sac.