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. Author manuscript; available in PMC: 2018 Feb 28.
Published in final edited form as: Nat Cell Biol. 2017 Aug 28;19(9):1116–1129. doi: 10.1038/ncb3596

Figure 1. Mitochondrial permeabilisation can engage necroptosis as a form of CICD.

Figure 1

(A) SVEC cells were treated with ABT-737 (10 μM) +/- Q-VD-OPh (10 μM) and immunoblotted for PARP and β-Actin. Representative images from three independent experiments.

(B) SVEC cells were treated for 72 h with ABT-737 (10 μM) +/- caspase inhibitor Q-VD-OPh (10 μM). For (B)(C)(D) and (E) cell viability was measured by flow-cytometry and PI exclusion. n=5 independent experiments.

(C) SVEC cells stably expressing LZRS empty vector (vector) or LZRS-MCL-1 were treated for 72 h with ABT-737 (10 μM) +/- Q-VD-OPh (10 μM). n=3 independent experiments; mean values ± S.E.M

(D) Control or RIPK3SH SVEC cells were treated for 72 h with TNF (20 ng/ml) +/- caspase inhibitor zVAD-FMK (50 μM) and/or RIPK1 inhibitor necrostatin-1 (30 μM). n=3 independent experiments; mean values ± S.E.M

(E) SVEC cells stably expressing pLKO empty vector (vector) or pLKO-shRIPK3 (RIPK3SH) were treated for 72 h with ABT-737 (10 μM) +/- caspase inhibitor Q-VD-OPh (10 μM) and/or RIPK1 inhibitor necrostatin-1 (30 μM). n=3 independent experiments; mean values ± S.E.M.

(F) BCL-xL dependent SVEC cells expressing empty vector (pLKO1) or pLKO-shAPAF-1 (APAF-1SH) were treated for 24 h with ABT-737 (10 μM) +/- caspase inhibitor Q-VD-OPh (30 μM). For (F)(H)(I)(J) cell death was measured using an IncuCyte imager, measuring SYTOX Green uptake. n=3 independent experiments; mean values ± S.E.M.

(G) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) +/- Q-VD-OPh (30 μM) then immunoblotted for indicated proteins. Representative images from three independent experiments.

(H) BCL-xL dependent SVEC cells stably expressing an empty vector (pLKO1) or pLKO1-shRIPK3 (RIPK3SH) were treated with ABT-737 (10 μM) in the presence of caspase inhibitor Q-VD-OPh (30 μM) and/or necrostatin-1 (30 μM). A representative time-point shown (16 h). n=5 independent experiments; mean values ± S.E.M.

(I) Control or MLKL-deleted BCL-xL dependent SVEC cells were treated with TNF (20 ng/ml) and zVAD-FMK (50 μM) +/- necrostatin-1 (30 μM). A representative time-point is shown (21 h). n=3 independent experiments; mean values ± S.E.M.

(J) Control (vectorCRISPR) or MLKL-deleted (MLKLCRISPR) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) together with Q-VD-OPh (30 μM) and/or necrostatin-1 (30 μM). A representative time-point is shown (21 h). n=3 independent experiments; mean values ± S.E.M.*p<0.05, **p<0.01, ***P<0.001; Holm-Sidak-corrected one way ANOVA (B), two-tailed unpaired t-test (C, D, E, H, I, J).