Extended Data Fig. 5: Intersection of the most pronounced positive regulators of CHOPNeon across genetic screens and validation of DELE1 and HRI as mediators of mitochondrial stress.
From: A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol
a, Data from Fig. 1a, b and Extended Data Fig. 1f, processed in the same way, except that genes that were identified as highly significant positive regulators (two-sided Fisher’s exact test, Padj < 10−5) with a mutation ratio lower than 0.25 in the CCCP dataset but not in either of the other two datasets are highlighted in red. b, Venn diagram showing the results of stringent filtering of positive CHOPNeon regulators from a (two-sided Fisher’s exact test, Padj < 10−5 and mutation ratio lower than 0.25) as intersections between all three of the genetic screens that were performed in this study. CCDC101 is also known as SGF29. c, HeLa cells, 293T cells, BJEH fibroblasts and N/TERT-1 keratinocytes were exposed to sgRNAs directed against the specified genes and pharmacologically stimulated for 9–12 h before immunoblotting (one representative experiment shown of n = 2 (HeLa and 293T) or n = 3 (BJEH and N/TERT-1) independent experiments). sgCTR, non-targeting control sgRNA. d, Clonal HAP1 knockout and stably reconstituted cells were treated as indicated (CCCP, tunicamycin and oligomycin (OM), 9 h; CDDO and GTPP, 11 h) and analysed by immunoblotting. e, HAP1 CHOPNeon cells of the indicated genotypes were treated for 9 h (CCCP, tunicamycin, CDDO) or 12 h (GTPP) and analysed by flow cytometry. Per genotype and treatment, the CHOPNeon signal was normalized to its DMSO control and statistical significance is indicated compared to identically treated wild-type cells (mean ± s.d. of n = 3 independent experiments; one-way ANOVA with Dunnett’s multiple comparisons correction). f, Wild-type, DELE1-deficient (clones #3 and #6) and stably reconstituted HAP1 CHOPNeon cells were transduced with lentiviral constructs that contain a non-targeting control sgRNA or an sgRNA directed against LONP1. CHOPNeon levels were analysed by flow cytometry. Per genotype, the CHOPNeon fluorescence detected in sgLONP1-treated cells is shown relative to the CHOPNeon fluorescence detected in the matching sgCTR-treated cells (n = 2 independent experiments). g, HeLa, 293T and HCT116 cells—either wild type or that were transiently exposed to an sgRNA directed against EIF2AK1 (HRI) or DELE1—were stimulated with CCCP for 1 h and the phosphorylation of eIF2α was analysed by immunoblotting (one representative experiment shown of two independent experiments).
