Extended Data Fig. 6: ZNF598^RING expression in human stem cells induces cytotoxicity but not through the ISR.

a, Immunoblot analysis of wild-type hiPSCi and hiPSC transduced with cDNA encoding HA-tagged wild-type (ZNF598^WT) or C29S/C32S (ZNF598^RING) ZNF598 (n = 2 biological replicates). b, Stalling readthrough of reporters containing an AAA-encoded stretch of twenty lysines (AAA₂₀) in control hiPSCi and hiPSCi transduced with ZNF598^WT or ZNF598^RING cDNA. The median fluorescence intensity for BFP and mOrange was quantified by flow cytometry ( > 20,000 cells/ analysis) and normalized to average values for the hiPSCi control (n = 3 biological replicates; P-values from unpaired two-tailed t-test). c, Global protein synthesis measurements by OP-Puromycin and AF647 detection. Median fluorescence intensity was quantified by flow cytometry ( > 10,000 cells/ analysis) and normalized to average values for the control (n = 6 biological replicates; p-values from unpaired two-tailed t-test). d, Lactate dehydrogenase (LDH) measurements in culture supernatants from wild type or ZNF598^RING-expressing hiPSCi two days after transduction. Values were normalized to supernatant from wild type hiPSCi (n = 4 technical replicates and 5 biological replicates; P-values from unpaired two-tailed t-test). e,f, Differential gene expression analysis in hiPSCi upon ZNF598 knockdown (sgZNF598) in comparison to a non-targeting sgRNA (sgControl), or in ZNF598^RING-expressing hiPSCi in comparison to untransduced hiPSCi (n = 2 biological replicates). (e) Heatmap of mRNAs differentially expressed in at least one context (two-sided Wald test; P-adj ≤ 0.05; n = 2289). (f) Heatmap of a subset from (e) showing genes associated with the ISR. g, Flow cytometry analysis of eIF2α (top) or p38 (bottom) phosphorylation upon ZNF598^RING expression in hiPSCi in comparison to cells transduced with a non-targeting sgRNA (sgControl; n = 2 biological replicates) on day 1-3 after transduction. h, Gene ontology (GO) enrichment in differentially expressed mRNAs from (e) using the “Biological Process” function in ClusterProfiler (one-tailed Fisher’s exact test with Benjamini-Hochberg correction, P-adj ≤ 0.01).

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