Fig 2: ER stress-induced cell death in apoptosome-compromised cells is accompanied by caspase activation. (A and B) Casp9+/+ and casp9−/− MEFs were treated for the indicated times with (A) 0.5 μM Tg or (B) 0.5 μg/ml of Tm and lysates immunoblotted for CASP9, CASP3, PARP, and actin. (C) Bax+/+ Bak1+/+ and bax−/− bak1−/− MEFs were treated with 1 μg/mL of Tm for indicated timepoints. Cell viability was analyzed by PI uptake. (D) Bax+/+ Bak1+/+ and bax−/− bak1−/− MEFs were treated with 1 μg/ml of Tm for the indicated times and lysates were immunoblotted for CASP9, CASP3, PARP and tubulin. (E) Bax+/+ Bak1+/+ and bax−/− bak1−/− MEFs were treated with 1 μg/ml of Tm for indicated times and CASP3-like activity was determined by DEVD-AMC hydrolysis. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD. (G) casp9−/− MEFs were treated with 0.5 μM Tg with or without Boc-D-FMK for the indicated times and lysates immunoblotted for PARP and cleaved CASP3. (H and I) casp9−/− MEFs were treated with (H) 0.5 μM Tg or (I) 0.5 μg/ml of Tm for the indicated times and lysates immunoblotted for cleaved CASP8.

Fig 3: Pro-CASP8 is recruited to a novel complex comprising of ATG5 and FADD in apoptosome-compromised cells exposed to sustained ER stress. casp9−/− MEFs were treated with a combination of 0.5 μg/ml of Tm for 72 h with 20 μM of Boc-D-FMK added for the last 48 h followed by immunoprecipitation with (A) a control IgG antibody or antibody specific for ATG5 or (B) a control IgG antibody or antibody specific for FADD. Immune complexes were analyzed by immunoblotting for ATG5, FADD, and pro-CASP8. IgG heavy chain band is marked as *. (C) bax−/− bak1−/− MEFs were treated with a combination of 0.5 μM of Tg for 48 h with 20 μM of Boc-D-FMK added for the last 24 h followed by immunoprecipitation with a control IgG antibody or antibody specific for FADD. Immune complexes were analyzed by immunoblotting for ATG5, FADD, and pro-CASP8. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD.

Fig 4: Defect of Ripk1D325A/D325A mice at E10.5 is mediated by TNFR1–RIPK3–caspase-8 signaling axis.(A) Genetic analysis of progeny from intercrossing Ripk1D325A/+ Casp8+/− mice. (B) Representative images of E10.5 and E12.5 embryos analyzed in (A). Scale bars, 1 mm. (C) Genetic analysis of offspring from intercrossing Ripk1D325A/+ Casp8−/− Mlkl−/− parents. (D) IF staining of E10.5 YS with anti-PECAM (red) and anti-Cl.CASP8 (green) antibodies. Scale bars, 50 μm. Images are representative of 4 embryos per genotype. See also S2 and S3 Figs. Cl.CASP8, cleaved caspase-8; E10.5, embryonic day 10.5; E12.5, embryonic day 12.5; EP, embryo proper; IF, immunofluorescence; PECAM, platelet endothelial cell adhesion molecule; Ripk1, receptor interacting serine/threonine kinase 1; TNFR1, tumor necrosis factor receptor-1; YS, yolk sac.

Fig 5: Exogenous expression of WT caspase-8, but not MT caspase-8 proteins, induces cell death.A HeLa-CASP8 KO cells engineered for DOX-inducible expression of WT caspase-8, MT caspase-8 proteins, or LacZ were treated in quadruplicate in the absence or presence of DOX (1 μg/mL) for varying lengths of time, followed by the performance of crystal violet (top panel) or MTT (bottom panel) assays. Data are expressed relative to no DOX treatment and one-way ANOVA was used to compare treatment groups. Columns represent the means from triplicate experiments; error bars represent the SEM. B In the left panel, cells were treated for 12 h in the absence or presence of DOX, followed by flow cytometric analysis of annexin V staining. Columns represent the average percentage of annexin V-positive cells from two independent experiments; error bars represent the SEM. Student’s t test was performed to compare groups with and without DOX induction. In the right panel, cells were treated in the absence or presence of DOX alone, or DOX plus z-VAD-FMK 30 μM) prior to analysis.