Figure EV1. Ubiquitylation of mitochondria is dependent on MOMP by BAX/BAK pores, but independent of caspase activity.

(A) U2OS EMPTY^CRISPR and BAX/BAK^CRISPR cells were treated with 10 μΜ ABT-737, 2 μΜ S63845 with or without 20 μΜ Q-VD-OPh. Cell death was determined using Sytox Green inclusion normalised to starting confluence. Graph is representative of three independent experiments and displays mean values ± s.e.m. (error bars) of technical triplicates. (B) Lysates from U2OS EMPTY^CRISPR and BAX/BAK^CRISPR cells were blotted for BAX, BAK and Actin. (C) SVEC4-10 EMPTY^CRISPR and BAX/BAK^CRISPR cells were treated for 1 h with 10 μΜ ABT-737, 10 μΜ S63845 and 30 μΜ Q-VD-OPh. Mitochondria were isolated using digitonin fractionation buffer and blotted for ubiquitin (UBCJ2), BAX, BAK, HSP60 and Actin. (D) SVEC4-10 cells were treated for 1 h with 10 μΜ ABT-737, 10 μΜ S63845 with or without 30 μΜ Q-VD-OPh. Mitochondria were isolated using digitonin fractionation buffer and lysates were blotted for ubiquitin (UBCJ2), HSP60 and actin. (E) U2OS cells were treated for 7 h with 10 μΜ ABT-737, 2 μΜ S63845 and 20 μΜ Q-VD-OPh with or without the addition of 10 μM MG-132. Lysates were blotted for ubiquitin (UBCJ2) and GAPDH. (F) U2OS cells were treated for 24 h with 10 μΜ ABT-737, 2 μΜ S63845 and 20 μΜ Q-VD-OPh with or without the addition of 10 μM MG-132. Mitochondrial depletion was assessed by blotting for several mitochondrial proteins and GAPDH. (G) U2OS cells were treated for 7 h with 10 μΜ ABT-737, 2 μΜ S63845 and 20 μΜ Q-VD-OPh with or without the addition of 1 μΜ TAK-243. Lysates were blotted for ubiquitin (UBCJ2) and GAPDH. (H) U2OS cells were treated for 24 h with 10 μΜ ABT-737, 2 μΜ S63845 and 20 μΜ Q-VD-OPh with or without the addition of 1 μΜ TAK-243. Mitochondrial depletion was assessed by blotting for several mitochondrial proteins and GAPDH. Data information: (C, D, E, F, G, H) blots are representative of three independent experiments.