Fig 2: ZBP1 and RIPK1 kinase activity regulate TRIFosome formation.a ZBP1 immunoprecipitation in B6 BMDMs stimulated as indicated and probed for RIPK1 and CASP8. b FADD and c ZBP1 immunoprecipitations in B6 and RIPK1 kinase-inactive BMDMs stimulated as indicated and probed for ZBP1, RIPK1, and CASP8. d, e FADD immunoprecipitation in B6, d Ripk3−/−Casp8−/−, or e Zbp1−/− BMDMs stimulated as indicated and probed for ZBP1, RIPK1, and CASP8. Data from western blots are representative of 3 or more biologically independent experiments. Source data for all experiments are provided as a Source data file.

Fig 3: Anti-necroptosis effects of compound 25in vitro. HT-29 cells were pretreated by compound 25 followed by incubation with TSZ (A), TCZ (B), TS (C) and TC (D) for 12, 24, 36 and 24 h, respectively. (E) L929 cells were pretreated with compound 25 followed by incubation with TZ for 9 h (∗∗∗P < 0.001 versus stimulators without compound 25). (F) HT-29 cells were pretreated with compound (1 μmol/L) followed by stimulation with TSZ at the indicated time points. Cells were lysed and immunoblotted with the indicated antibodies. (G) HT-29 cells were pretreated with compound (0, 0.01, 0.1, 1 μmol/L) and then stimulated with TSZ for 4 h. Cells were lysed and immunoblotted with the indicated antibodies. (H) HT-29 cells were treated with DMSO or compound 25 (1 μmol/L) for 6 h. The cell lysates were immunoprecipitated with anti-RIP1 antibody (IP: RIP1) and analyzed by Western blotting with an anti-RIP3 antibody (IB: RIP3). (I) DARTS analysis for 25. HT-29 cell lysates were incubated with or without 25 (50 μmol/L) for 1 h and then digested with 0.1% Pronase for 30 min. Cells were lysed and immunoblotted with the indicated antibodies.

Fig 4: RI-962 protected cells from TSZ-induced necroptosis.a–d Dose–response curves of RI-962 (n = 3) and GSK3145095 (n = 2) in TSZ-induced HT29, L929, J774A.1, and U937 cell necroptosis models. Necroptosis of HT29, J774A.1 and U937 cells was induced by TNFα (10 ng/mL), Smac-mimetic (100 nM), and Z-VAD-FMK (40 μM). Necroptosis of L929 cells was induced by TNFα (10 ng/mL) and Z-VAD-FMK (40 μM). Cell viability was measured by CCK8 staining after 24 h of inducing necroptosis. Data are presented as mean ± standard deviation. e Dual staining with CytoCalcein Violet 450 and 7-AAD in HT29 cells with/without RI-962 and GSK3145095 treatment after TSZ-induced necroptosis for 24 h. f Statistic percentage of dead cells in the dual staining assay (n = 4). Data are presented as mean ± standard deviation, **p-value < 0.01, ***p-value < 0.001 by two-tailed unpaired Student’s t-test. g, h Knockout of RIPK1 desensitizes HT29 cells to TSZ-induced necroptosis. Cell viability was measured by CCK8 staining after 24 h of inducing necroptosis (n = 3). Data are presented as mean ± standard deviation. i RI-962 inhibited the phosphorylation of RIPK1, RIPK3, and MLKL in HT29 cells. β-actin was used as an internal control. Molecular weight (Mw) markers are shown at right. The experiment was performed three times with similar results. j Knockout of RIPK1 inhibited the phosphorylation of RIPK3 and MLKL, and compound RI-962 had the same effect. β-actin was used as an internal control. Molecular weight (Mw) markers are shown at right. The experiment was performed three times with similar results. Source data are provided as a Source Data file.

Fig 5: ZBP1 promotes CASP8-mediated cell death and IL-1β release.a Schematic of protein domains in TRIF, RIPK1, and ZBP1. b, c Cell death over time in indicated BMDMs stimulated with LPS/5z7 as measured by (b) propidium iodide (PI) incorporation or (c) LDH release. d Cell death over time as measured by PI incorporation in B6 and Zbp1−/− BMDMs treated as indicated. e Full-length and cleaved products of indicated caspases and GSDMD from whole-cell lysates (WCL) or precipitated from the supernatant (Sup.) of B6 or Zbp1−/− BMDMs stimulated with LPS/5z7 for 1–6 h. f Levels of total, pRIPK1 (S166), and pRIPK1 (S321) in B6 or Zbp1−/− BMDMs stimulated with LPS or LPS/5z7 for 1–3 h. g Full-length and cleaved products of CASP1 and IL-1β from whole-cell lysates of B6 and Zbp1−/− BMDMs stimulated as indicated for 1–3 h. h IL-1β release after 6 h and (i) percentage of ASC+ cells after 2 h in B6, Zbp1−/−, and Ripk3−/−Casp8−/− BMDMs stimulated as indicated. Data from cell death assays and western blots are representative of 3 or more biologically independent experiments, cell death data are presented as the mean ± SD of triplicate wells, n = 10,000 cells examined in three individual wells. IL-1β release, ASC percentage, and qPCR data are presented as the mean ± SD for triplicate wells from n = 4 biologically independent experiments. Two-way analysis of variance (ANOVA) was used for comparison between groups. Source data for all experiments are provided as a Source data file. See also Supplementary Figs. 2 and 3.