Figure 1. The eIF2α kinase HRI relays mitochondrial stress to ATF4.

(a) ATF4 translational reporter including the upstream open reading frames (uORFs) of the ATF4 5’ untranslated region (5’UTR) followed by mApple replacing the ATF4 coding sequence. Transcription of the reporter and an EGFP transcriptional control is driven by the CMV promoter.

(b,c) Reporter cells expressing either single sgRNAs (b) or triple sgRNAs (c) targeting the indicated eIF2α kinases were exposed to 1.25 ng/mL oligomycin for 16 h before measuring reporter levels by flow cytometry (mean ± s.d., n = 3 culture wells).

(d) Immunoblot of endogenous ATF4. Cells expressing a non-targeting control sgRNA (NTC) or sgRNAs targeting HRI were treated with 1.25 ng/mL of oligomycin for 16 h where indicated. Left, representative blot; Right, quantification of n = 2 blots.

(e) Expression of the heme-induced gene HO-1 after 24-hour incubation with the indicated concentrations of hemin, measured by qPCR (n = 3 technical replicates).

(f) Heme supplementation does not abolish ATF4 induction. Reporter levels in cells were treated with the indicated concentrations of hemin for 24 h before a 16 h treatment with 1.25 ng/mL oligomycin in the presence or absence of hemin (mean ± s.d., n = 3 culture wells).

(g) Oligomycin treatment used in this study does not induce reactive oxygen species (ROS). Cells were treated with 1.25 ng/mL oligomycin or 40 nM rotenone for 16 h and ROS levels were quantified by flow cytometry using the CellROX reagent (mean ± s.d., n = 3 culture wells).

(h) Model.