a, HEK293T cells were treated with 50 μg ml−1 doxycycline or 1.25 ng ml−1 oligomycin for 16 h, and transcript levels were compared to untreated cells using RNA sequencing for n = 2 (doxycycline) or n = 3 (oligomycin) independent experiments. Differentially expressed genes and P values were determined as described in the Methods (full datasets in Supplementary Tables 1, 2). Significantly induced genes (P adj < 0.05), with at least a twofold increase in treated over untreated conditions, are included in the analysis. Enrichment analysis for targets of transcription factors that are annotated in the TRRUST database57 (statistical analysis described in the Methods) detected ATF4 as the only significant transcription factor (P adj < 0.05) for both treatments. Genes induced by both treatments are listed, as well as genes annotated as ATF4 targets in TRRUST (dark green dots) or a previous study1 (light green dots). CARS is also known as CARS1. b, Quantification of the knockdown efficiency of sgRNAs that target the eIF2α kinases by qPCR (n = 3 technical replicates). For HRI, two independent sgRNAs (1 and 2) were characterized. WT, wild type. c, d, Validation of the reporter cell line using the endoplasmic-reticulum stressor thapsigargin. Reporter cells expressing either individual (c) or triple (d) sgRNAs that target the indicated eIF2α kinases were exposed to 75 nM thapsigargin for 8 h before measuring reporter levels by flow cytometry. The induction of the ATF4 reporter by thapsigargin is blocked by knockdown of PERK. The fold change in reporter levels was quantified as in Fig. 1b (mean ± s.d., n = 3 culture wells). e, Pharmacological inhibition of mitochondrial function using the mitochondrial ribosome inhibitor doxycycline, the electron transport chain inhibitors antimycin A and rotenone and the ATP synthase inhibitor oligomycin induces the ATF4 reporter. Reporter cells were exposed to the indicated treatments for 16 h before measuring reporter levels by flow cytometry. The fold change in reporter levels was quantified as in Fig. 1b (mean ± s.d., n = 3 culture wells). f, The ATF4 reporter is induced in cells with CRISPRi knockdown of factors that are required for mitochondrial protein homeostasis (HSPD1 and LONP1) and mitochondrial ribosomal proteins (MRPL17 and MRPL22) (red), compared to wild-type cells (blue). Similar results in more than three independent experiments. g, DELE1 and HRI are not required to trigger the ISR in response to endoplasmic-reticulum stress. Reporter cells expressing non-targeting control sgRNAs or sgRNAs that target HRI or DELE1 were exposed to 75 nM thapsigargin for 8 h before measuring reporter levels by flow cytometry. The fold change in reporter levels (mean ± s.d., n = 3 culture wells) is the ratio of median fluorescence values for thapsigargin-treated over untreated samples.