Extended Data Fig. 11: Cellular fitness and mitochondrial function in cells in which the DELE1–HRI–eIF2α axis is perturbed.
From: A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol
a, HAP1 cells were infected with lentivirus expressing TagRFP and the indicated sgRNAs. A 1:1 mixture of infected and uninfected cells was treated for 48 h with 5 μM CCCP or DMSO and analysed by flow cytometry. The abundance of RFP+ sgRNA-containing cells after CCCP treatment was compared with the respective DMSO-treated sample bearing the same sgRNA and normalized to a non-targeting control sgRNA. Statistical significance was assessed compared to a second non-targeting control sgRNA using one-way ANOVA with Dunnett’s multiple comparisons correction (mean ± s.d. of n = 5 independent experiments). b, c, HAP1 cells of the indicated genotypes were treated as indicated for 48 h and cell survival was visualized using crystal violet (one representative experiment shown of three independent experiments). d–j, Wild-type and DELE1-knockout HeLa cells stably expressing the indicated cDNAs were subjected to different mitochondrial stressors and the oxygen consumption rate (OCR) was monitored. Within the same clonal background, no significant difference between DELE1-deficient and DELE1-proficient cells could be observed in non-mitochondrial oxygen consumption (e), basal respiration (f), maximal respiration (g), proton leak (h), ATP production (i) and spare respiratory capacity (j). Mean ± s.d. of n = 4 independent culture wells from one representative experiment of three independent experiments is shown; statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons correction. k, l, Wild-type, MFN2-knockout and stably reconstituted HAP1 cells were lentivirally transduced with a construct encoding TagRFP together with a non-targeting control sgRNA or an sgRNA directed against EIF2AK1 (HRI) (k) or DELE1 (l) and the fraction of RFP+ cells was monitored over time. The abundance of sgDELE1 or sgHRI-containing cells was normalized to non-targeting sgRNA and to time 0 (mean ± s.d. of n = 3 biologically independent, separately infected and cultured wells per sgRNA and genotype; one-way ANOVA with Dunnett’s multiple comparisons correction; one representative experiment shown of two independent experiments). m, SH-SY5Y cells were exposed to the indicated sgRNAs and treated as in Extended Data Fig. 5c (one representative experiment shown of four independent experiments). n, o, Processing and cellular localization of DELE1–HA in SH-SY5Y cells, as in Extended Data Fig. 6k, o. MFN2–Flag–mNeon served as a control (one representative experiment shown of two independent experiments). Scale bars, 10 μm.
