Extended Data Fig. 6: DELE1 operates upstream of HRI, is cleaved and accumulates in the cytosol across different types of mitochondrial perturbation and different cellular systems.
From: A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol
a, b, HeLa wild-type, knockout and stably reconstituted cells were treated for 1 h (phosphorylated eIF2α) or 9 h (CHOP) with CCCP. Cells were analysed by immunoblotting (a) and the levels of phosphorylated eIF2α were quantified relative to ACTB (b; mean ± s.d. of n = 5 independent experiments; for each genotype, CCCP treatment was compared to DMSO using a paired two-tailed Student’s t-test). c, d, Subcellular localization of HA-tagged DELE1 or DELE1(∆MTS) in HeLa cells analysed by confocal microscopy. TRAP1 staining identifies mitochondria and nuclei were visualized with DAPI. e, Top, clonal DELE1-knockout HAP1 CHOPNeon cells were transiently transfected with full-length DELE1 or truncation mutants that lack the first 101 (DELE1(ΔMTS(N101))) or 115 (DELE1(ΔMTS(N115))) amino acids as indicated. Bottom, transfected cells were treated with CCCP or DMSO for 9 h and analysed by flow cytometry as in Fig. 2a (mean ± s.d. of n = 3 independent biological samples; two-way ANOVA with Tukey’s multiple comparisons correction; one representative experiment shown of three independent experiments). f, HeLa cells were transfected with DELE1-HA, treated with CCCP alone or CCCP and cycloheximide (CHX) as indicated and analysed for subcellular localization of DELE1. Mitochondria were stained with MitoTracker and nuclei were visualized using DAPI. g, CRISPR engineering of the DELE1 locus in 293T cells (introducing a C-terminal in-frame fusion with a triple HA tag), followed by treatment with CCCP alone or CCCP and cycloheximide and analysis by immunoblotting (one representative experiment shown of four independent experiments). h, Wild-type and HRI-knockout HAP1 cells were transfected with DELE1-HA and treated as indicated. The fate of the DELE1–HA protein was analysed by immunoblotting (n = 5 (HRI) or 2 (wild type) biologically independent clones). i, j, HCT116 (i) and HAP1 (j) cells were transfected with DELE1-HA, treated with the specified compounds and analysed for DELE1–HA cleavage and CHOP induction by immunoblotting (one representative experiment shown of two independent experiments). k, N/TERT-1 keratinocytes, hTERT+ (BJEH) fibroblasts and primary fibroblasts were transduced with DELE1–HA, stimulated for 4 h as indicated and assayed by immunoblotting. l, Wild-type 293T and HAP1 cells were transfected with DELE1-HA and treated with CCCP for 4 h. The localization of DELE1–HA was analysed as in Fig. 2d (one representative experiment shown of two independent experiments). m–o, Cells from k were stimulated as indicated for 4 h and the localization of DELE1–HA and MFN2 were analysed by confocal microscopy. TRAP1 staining (m), MitoTracker (n, o) and DAPI (o) were used to visualize cellular substructures (one representative experiment shown of two independent experiments). Scale bars, 10 μm (c, d, f, l–o).
