Fig. 2: iRhoms use the intrinsic apoptotic pathway under ER stress.

a Schematic showing the intrinsic and extrinsic cell death pathways regulated by caspases. b Cell lysates of WT and iRhom1/2 DKO MEFs after exposure to tunicamycin (0.5 µg/ml) for the indicated times were analysed by immunoblotting with indicated antibodies. c Cell lysates of WT, iRhom1/2 DKO and iRhom1/2 DKO MEFs stably reconstituted with iRhom1-3xFLAG and iRhom2-3xHA after exposure to tunicamycin (0.5 µg/ml or 1 µg/ml, 18 h) were analysed by immunoblotting with indicated antibodies. White triangle symbols denote major bands for iRhoms. d Mitochondrial membrane depolarisation in WT and iRhom1/2 DKO MEFs was measured using TMRE dye (250 nM) 18 h after treatment with tunicamycin (0.25 µg/ml or 0.5 µg/ml) or brefeldin A (1 µg/ml). FCCP (50 µM) was added for 15 min prior to measurement (n = 4, biologically independent experiments). Data are expressed as mean ± SEM. Two-way ANOVA (Sidak’s), **p < 0.01 ****p < 0.0001 and ns = not significant. e Cell lysates of WT and iRhom1/2 DKO MEFs exposed to tunicamycin (0.5 µg/ml, 18 h) in the presence of ADAM protease inhibitors GI254023X (5 µM; specific to ADAM10) and GW280264X (5 µM; inhibits both ADAM10 and ADAM17) were analysed by immunoblotting with indicated antibodies. Bar chart shows quantification of cleaved caspase-3 relative to WT+DMSO as mean ± SEM (n = 3, biologically independent experiments). Two-way ANOVA (Sidak’s), ***p < 0.001. f Cell lysates of WT and iRhom1/2 DKO MEFs treated for 18 h with cycloheximide (CHX 10 µg/ml) and/or TNF (2.5 ng/ml or 5 ng/ml) were analysed by immunoblotting with indicated antibodies. *Denotes unspecific band. Immunoblotting data are representative of 2–3 biologically independent experiments. Source data are provided as a Source data file.