Extended Data Fig. 6: Examination of the mitochondrial stress response with a broad range of mitochondrial toxins and in non-HEK293T cells.

a, Immunoblot of ATF4 in wild-type HEK293T cells and those treated with sgRNAs targeting DELE1, HRI or OMA1 under different mitochondrial stress conditions. Cells were left untreated or treated with 40 nM antimycin A (AA), 40 nM rotenone (rot), 50 μg ml⁻¹ doxycycline (dox) or 5 μM CCCP for 2 and 4 h. Left, representative blots. Right, ATF4 levels were quantified and normalized to β-actin (mean ± s.d., n = 2 blots). b, A broad range of mitochondrial toxins stimulates the accumulation of DELE1_S. Cells stably expressing DELE1-mClover were untreated or treated with a panel of mitochondrial toxins for 16 h (see Methods for details) and subjected to western blotting with antibodies detecting DELE1-mClover, ATF4 and actin. Top, representative blot. Middle, bottom, ATF4, DELE1_L-mClover and DELE1_S-mClover levels were quantified (mean ± s.d., n = 2 blots). c, Subcellular localization of DELE1_L and DELE1_S in cells that were treated with a broad range of mitochondrial toxins. Biochemical fractionation of cells stably expressing DELE1-mClover that were either treated with different mitochondrial toxins as indicated for 16 h or left untreated. β-actin and LONP1 were probed as markers for cytosol and mitochondria, respectively. Similar results in n = 2 independent experiments. d, Examination of OPA1 cleavage in cells treated with a broad range of mitochondrial toxins. Similar results in n = 2 independent experiments. e, Immunoblot of ATF4 in wild-type cells and those treated with sgRNAs targeting DELE1, HRI or OMA1 in the WTC11 human iPSC line. Cells were left untreated or treated with 1.25 ng ml⁻¹ oligomycin for 4 h. Similar results in n = 2 independent experiments. f, Immunoblot of ATF4 in wild-type cells and those treated with sgRNAs targeting DELE1, HRI or OMA1 in the human HeLa cell line. Cells were left untreated or treated with 1.25 ng ml⁻¹ oligomycin for 2 and 4 h. Similar results in n = 2 technical replicates. For gel source data, see Supplementary Fig. 1.