Fig 2: 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 3: 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 4: Impact of WT or MT caspase-8 expression on tumor immune microenvironment in vivo.MOC1-CASP8 KO cells engineered for DOX-inducible expression of WT caspase-8 or the D303G caspase-8 mutant were inoculated into the flanks of wild-type C57BL/6 mice. On day 28 (see Supplementary Fig. 8), mice were randomized to receive DOX (in drinking water) or no DOX. Tumors were harvested after 18 days of treatment and analyzed by flow cytometry for lymphoid (A) or myeloid (B) cell populations. In the DOX treatment groups, only tumors demonstrating DOX induction of the FLAG-tagged WT or MT caspase-8 (Supplementary Fig. 7) were analyzed. Error bars indicate SEM. Student’s t tests were performed to compare WT with DOX vs. WT without DOX; D303G with DOX vs. D303G without DOX, and WT with DOX vs. D303G with DOX.

Fig 5: Staining of actin filaments (green) of control and Casp8 -/- cells after doxorubicin treatment compared to untreated controls (A–D). Activation of caspase-3 (green) in control and Casp8 -/- cells after doxorubicin treatment compared to untreated controls (E–H). Nuclei of doxorubicin treated samples are red, untreated samples are counterstained by DAPI (blue). Scale bar = 100 µm. Bioluminescent measurement of caspase3/7 (I) and caspase-6 (J) activity in Casp8 -/- and control cells after doxorubicin treatment compared to untreated controls. Different letters indicate statistically significant differences (p < 0.001), as estimated using ANOVA. Dox doxorubicin, UT untreated.