Fig 1: Loss of E3 ligase activity alters the TNF‐response in MEFs MEFs were treated with TNF for the indicated times. Cell lysates were analysed by WB (*indicates non‐specific bands). WB of cIAP1Wt/Wt MEFs treated with TNF for the indicated time points.Cells were pre‐treated with 20 μM birinapant 3 h prior to TNF stimulation as indicated. Cell lysates were analysed by WB.IncuCyte analysis to determine % cell death in MEFs after 6‐h treatment as indicated. Bars represent mean ± SEM. P‐values were calculated using one‐way ANOVA followed by Bonferroni post analysis. **P < 0.01 and ****P < 0.0001. MEFs were treated with combinations of TNF (100 ng/ml), IDN (5 μM), Nec‐1S (20 μM) and birinapant (20 μM) for 6 h; n = 3; technical replicate shown.Representative images of spleen and colon sections from mice at 2 weeks of age. Black arrowheads indicate areas of apoptotic cells. Scale bars: 100 μm. Sections were stained with haematoxylin and eosin (H&E), cleaved caspase‐3 (CC3), or Ki67.
Fig 2: Loss of cIAP E3 ligase activity leads to NIK accumulation and activation of non‐canonical NFκB signalling in vivo Representative pictures of Tnfr1 KO/KO /Ripk1 D138N/D138N /cIAP1/2 Wt/Wt and Tnfr1 KO/KO /Ripk1 D138N/D138N /cIap1/2 MutR/MutR mice at 2 weeks of age.Body weight, liver weight/body weight ratios (LW/BW), spleen weight/body weight ratios (SpW/BW) from age‐matched mice of the indicated genotypes.Representative images of liver, spleen, colon and ileum sections from Tnfr1 KO/KO /Ripk1 D138N/D138N /cIAP1/2 Wt/Wt mice at 2 weeks of age. Scale bars: 50 μm. Sections were stained with Hematoxylin and Eosin (H&E), cleaved caspase‐3 (CC3), Ki67, or F4/80.IncuCyte analysis to determine cell death in MEFs after 48 h of TNF treatment (100 ng/ml).WB of liver lysates from young mice with the indicated genotypes (2 mice per genotype).WB of liver lysates from aged mice with the indicated genotypes (1 mouse per genotype).Gene expression analysis of MEFs treated with NIK inhibitor (5 μM) for 24 h, relative mRNA expression of indicated genes was measured by qPCR. Data information: In (B), dots represent individual mice and lines the means. In (D) and (G), bars represent mean ± SEM of three technical replicates. P‐values were calculated using one‐way (B and D) or two‐way (G) ANOVA followed by Bonferroni postanalysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns, not significant.
Fig 3: Hyperinflammatory phenotype in the aged Tnfr1 KO/KO /Ripk1 D138N/D138N /cIap1/2 MutR/MutR mice Representative images of liver and ileum sections from mice with the indicated genotypes at 24 weeks of age. Scale bars: 50 μm (liver) and 100 μm (ileum).Relative mRNA expression of the indicated genes from liver and ileum isolated from mice measured by qPCR. Bars represent mean of three mice per genotype ± SEM. P‐values were calculated using two‐way ANOVA followed by Bonferroni postanalysis. *P < 0.05; ***P < 0.001 and ns, not significant.Schematic illustration of cIAP‐mediated control of TNFR1‐induced cell death. The ubiquitin ligase activity of cIAP1/2 controls TNF‐induced RIPK1‐mediated apoptosis/necroptosis and provides the molecular platform for the activation of NFκB in response to TNF. Activation of NFκB survival signalling inhibits TNF‐induced TRADD‐mediated apoptosis. cIAP‐mediated ubiquitylation further controls TNF‐induced RIPK3‐dependent and RIPK1‐independent necroptosis. The underlying molecular mechanism remains undetermined. Source data are available online for this figure.
Fig 4: Role of cIAP1/2 E3 ligase activity in the apoptotic and necroptotic machinery independent of RIPK1 kinase activity IncuCyte analysis to determine % cell death in Wt MEFs treated with TNF (100 ng/ml), TNF + GSK (5 μM), TNF + Nec‐1S (20 μM), TNF + IDN (5 μM), or TNF + GSK + IDN at indicated time points.Representative IncuCyte images of dying MEFs (DRAQ‐7‐positive red cells) in Figs 3A and C, and EV3E after treatment with TNF, TNF + GSK, TNF + IDN, or TNF + IDN + GSK after indicated time points.Cell lysates from MEFs transfected with the respective siRNA for 24 h were analysed by WB.IncuCyte analysis to determine % cell death in cIAP1/2MutR/RIPK1D138N MEFs transfected with the respective siRNA and treated with TNF (100 ng/ml) for 30 h.IncuCyte analysis to determine % cell death in MEFs treated with TNF (100 ng/ml) and GSK (5 μM) at indicated time points.MEFs were treated with FLAG‐TNF (1 μg/ml) for the indicated times. TNFR1 protein complex was purified and analysed by WB.IncuCyte analysis to determine % cell death in cIAP1/2MutR/RIPK1D138N MEFs transfected with the respective siRNA and treated with TNF (100 ng/ml) for 30 h. Caspase‐3/7 (Casp‐3/7) activity was measured by IncuCyte analysis. Fluorescence signal of CellEvent Casp‐3/7‐positive cells was quantified and normalised to cell number. Data information: In (A) and (E), data are represented as mean ± SEM of three technical replicates. P‐values in (E) were calculated by repeated measures one‐way ANOVA with Bonferroni's postanalysis. Bars in (D) and (G) represent mean ± SEM of three technical replicates. P‐values were determined by one‐way ANOVA with Bonferroni's postanalysis. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 5: TNF induces apoptosis and necroptosis in cIAP1/2MutR/RIPK1D138N cells without involving RIPK1 kinase activity IncuCyte analysis to determine % cell death in MEFs treated with TNF (100 ng/ml) and TNF + Nec‐1S (20 μM) at indicated time points.MEFs were treated with TNF (100 ng/ml) and Nec‐1S (20 μM) for the indicated times. Cell lysates were analysed by WB.IncuCyte analysis to determine % cell death of MEFs treated with TNF (100 ng/ml) and IDN‐6556 (IDN) (5 μM) and TNF, IDN and GSK‐872 (GSK) (5 μM), at indicated time points.MEFs were treated with TNF/IDN for 4 h. Cell lysates were analysed by WB.Representative images of liver and ileum sections from mice at 2 weeks of age. Black arrowheads indicate cells expressing phosphorylated RIPK3. Scale bars: 50 μm.Kaplan–Meier survival curves of Ripk3 KO/KO /Ripk1 D138N/D138N /cIAP1/2 Wt/Wt (n = 4) and Ripk3 KO/KO /Ripk1 D138N/D138N /cIap1 MutR/MutR /cIap2 MutR/MutR (n = 5) mice. P‐values were calculated with a log‐rank (Mantel–Cox) test.Body weight, liver weight/body weight ratios (LW/BW), spleen weight/body weight ratios (SpW/BW) from age‐matched mice of the indicated genotypes. Data information: In (A) and (C), data is represented as mean of three technical replicates ± SEM. P‐values were calculated by repeated measures one‐way ANOVA with Bonferroni's post analysis. Dots in (G) represent individual mice and lines the means. P‐values calculated by unpaired Student's t‐test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns, not significant. Source data are available online for this figure.
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