Extended Data Fig. 10. Stress response silencing restores cell survival.

a. Pharmacological stress response silencing in cells lacking UBR4 or KCMF1 through ISRIB. Wildtype, ΔUBR4, or ΔKCMF1 cells were treated with 5 μΜ sodium arsenite for 16 h and, as indicated, ISRIB. ATF4 levels were monitored by Western blotting. Similar results in n = 2 independent experiments. b. ISRIB inhibits stress response activation in cells that were lacking UBR4 or KCMF1 and were treated with antimycin A (0.6 μΜ) for 16 h. Similar results in n = 2 independent experiments. c. ISRIB inhibits stress response activation in cells that were lacking UBR4 or KCMF1 and were treated with BtdCPU (5 μΜ) for 8 h. Similar results in n = 2 independent experiments. d. ISRIB does not restore mitochondrial protein import in cells depleted of TIMM13. Import was measured upon GFP reconstitution by flow cytometry, as described above. Experiment was validated using the alternative mitochondrial import substrate HMT2-GFP11. e. ISRIB rescues ISR activation in human embryonic stem cells. As indicated, UBR4 was depleted by sgRNAs. Sodium arsenite (1.25 μΜ) and/or ISRIB were added for 8 h and ATF4 activation was monitored by Western blotting. Similar results in n = 2 independent experiments. f. Pharmacological silencing of the ISR with ISRIB rescues the synthetic lethality between UBR4 deletion and chemical mitochondrial stressors. Cell competition assays were performed as described above. Some competitions were performed at the same time as for Fig. 1d and are therefore re-reshown from Fig. 1d. g. Pharmacological silencing of the ISR with ISRIB rescues the cells depleted of the disease gene TIMM8A. Cell competition assays were performed as described above. For gel source data, see Supplementary Fig. 1.