Fig 2: cIAP1 and LUBAC facilitate ZBP1-induced inflammatory signaling ANF-?B activity in HEK293FT/RIPK3-2xFV cells transfected with siRNA targeting HOIP (siHOIP) or a mismatch sequence (siMM), and 48 h later transfected with dual luciferase reporters and ZBP1 or GFP (control). Luciferase reporter activities were measured 24 h after transfection and normalized to the siMM+ZBP1 samples. Data are plotted as mean with S.E.M. (n = 4 biological replicates). A Welch's t-test was used to test for the statistical difference between indicated conditions. ****P < 0.0001. HOIP knockdown and ZBP1 expression levels were analyzed by Western blotting. Blots are representative of three biological replicates.BNF-?B activity in HEK293FT/RIPK3-2xFV cells treated with 0 or 100 nM CpA and transfected with dual luciferase reporters and ZBP1 or EV. Luciferase reporter activities were measured 24 h after transfection and normalized to the 0 nM CpA + ZBP1 sample. Data are plotted as mean with S.E.M. (n = 3 biological replicates). A Welch's t-test was used to test for the statistical difference between indicated conditions. ****P < 0.0001.CCytokine concentration in the culture media of HT29/TO-ZBP1WT cells treated with 0 or 100 nM CpA and 0 or 50 ng/ml Dox as indicated for 24 h. Data are plotted as mean with S.E.M. (n = 3 biological replicates). Unpaired t-tests were used to test for the statistical difference between indicated conditions. **P = 0.0049; *P = 0.0394.D–FWestern blot analysis of anti-FLAG (ZBP1) immunoprecipitation from HT29 or HT29/TO-ZBP1WT or HT29/RIPK1-KO clone aA3/TO-ZBP1WT cells treated with 0 or 500 ng/ml Dox for 16 h. Asterisk indicates antibody heavy chain signal in IP samples. Blots are representative of at least (D) two and (E–F) three biological replicates. Source data are available online for this figure.

Fig 3: RIPK3 oligomerization induces ubiquitination-mediated inflammatory signaling ASchematic illustration of the chemical-inducible RIPK3 oligomerization system.BWestern blot analysis of HCT116/RIPK3-2xFV cells (FL and ?C) treated with 100 nM dimerizer. Blots are representative of two biological replicates.CTime courses of mRNA levels of CXCL8 and CXCL1 in HCT116/RIPK3-2xFV cells (FL and ?C) in response to treatment with 100 nM dimerizer. Data are plotted as mean with S.E.M. (n = 3 biological replicates). Two-way ANOVA and Tukey's multiple comparisons test were used to test for statistical differences between 0 h and indicated time points in FL cells. n.s. = not significant (P = 0.1360); **P = 0.0046 for FL 2 h, P = 0.0012 for FL 3 h; ****P < 0.0001; ***P = 0.0008.DCXCL8 concentration in the culture media of HCT116/RIPK3-2xFV cells (WT and RHIMmut) treated with 100 nM dimerizer for 24 h. In RIPK3-RHIMmut three key residues, VQV, of the RHIM region was changed to AAAA. Data are plotted as mean with S.E.M. (n = 5 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated time points. **P = 0.0011; n.s. = not significant (P = 0.9301). Inset: Western blot analysis to determine RIPK3 expression levels in one biological replicate. Line indicates that image was cut and spliced to remove nonrelevant lanes from the scanned blot.ERelative viability of HCT116/RIPK3-2xFV cells treated with 0 or 100 nM dimerizer for 24 h. Data are presented as mean with S.E.M. (n = 3 biological replicates). A Welch's t-test was used to test for statistical differences between indicated conditions. n.s. = not significant (P = 0.2294).FCell death analysis of HCT116/RIPK3-2xFV cells treated with 0 or 100 nM dimerizer or TSZ (100 nM CpA, 20 µM zVAD and 1 ng/ml TNF) for 24 h using SytoxGreen to stain dead cells. Data are plotted as mean with S.E.M. (n = 3 biological replicates). One-way ANOVA and Sidak's multiple comparisons test were used to test for statistical difference between indicated conditions. n.s., not significant (P = 0.1032); **P = 0.0014.G, HRelative mRNA levels of CXCL8 and CXCL1 in HCT116/RIPK3-2xFV cells pretreated with DMSO, TAK1 inhibitor, or IKK inhibitors for 1 h or 100 nM CpA for 0.5 h before treatment with 0 or 100 nM dimerizer for 3 h. Data are presented as mean with S.E.M. (n = 3 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated conditions and DMSO+dimerizer–treated condition. ****P < 0.0001. (H) Cell lysates were analyzed by Western blotting for cIAP1 levels. Blots are representative of two biological replicates.ICXCL8 concentration in the culture media of HCT116/RIPK3-2xFV cells pretreated with 100 nM CpA for 1 h before treatment with 0 or 100 nM dimerizer for 24 h. Data are presented as mean with S.E.M. (n = 3 biological replicates). An unpaired t-test was used to test for the statistical difference between indicated conditions. **P = 0.0042.JRelative CXCL8 mRNA levels in WT or HOIP-knockout HCT116/RIPK3-2xFV cells treated with 0 or 100 nM dimerizer for 3 h. Data are plotted as mean with S.E.M. (n = 3 biological replicates). A Welch's t-test was used to test for the statistical difference as indicated. ***P = 0.0010. Cell lysates were loaded for Western blot analysis.KRelative CXCL8 mRNA levels in HCT116/Tet-On-GFP-K63-SUB/RIPK3-2xFV, HCT116/Tet-On-GFP-M1-SUB/RIPK3-2xFV and HCT116/Tet-On-GFP/RIPK3-2xFV cells treated with 0 or 100 ng/ml Dox for 48 h before stimulated with 0 or 100 nM dimerizer for 3 h. Data are presented as mean with S.E.M. (n = 4 biological replicates). Brown-Forsythe and Welch ANOVA tests and Dunnet's T3 multiple comparisons test were used to test for statistical differences between indicated conditions. n.s. = not significant (P = 0.9997); **P = 0.0030; *P = 0.0366. Cells were analyzed by Western blotting for the inducible-expression levels of GFP (control) and GFP-SUBs. Blots are representative of three biological replicates.LRelative chemokine mRNA levels in HCT116/RIPK3-2xFV cells pretreated with 0 or 20 µM zVAD in combination with DMSO, 10 µM Nec1s, 10 µM GSK'872, or 1 µM NSA for 1 h, followed by treatment with 0 or 100 nM dimerizer for 3 h. Data are plotted as mean with S.E.M. (n = 3 biological replicates). ****P < 0.0001. One-way ANOVA and Sidak's multiple comparison test were used to test for statistical differences between indicated conditions.MRelative CXCL8 mRNA levels in HCT116/RIPK3-2xFV cells stably knocked down against mismatch (shMM) or two different sites of caspase-8 (shCASP8-A and shCASP8-B), pretreated with DMSO, 10 µM Nec1s, 10 µM GSK'872, or 1 µM NSA for 1 h followed by treatment with 0 or 100 nM dimerizer for 3 h. Data are plotted as mean with S.E.M. (n = 3 biological replicates). Two-way ANOVA and Tukey's multiple comparison tests were used to test for statistical differences between indicated conditions. ***P = 0.0001; **P = 0.0035. Cell lysates from one biological replicate were analyzed by Western blotting to determine caspase-8 levels. Source data are available online for this figure.