Fig. 1: USP37 loss protects cells from topoisomerase poisons- and replication stress-associated DNA damage.
From: USP37 prevents premature disassembly of stressed replisomes by TRAIP
a Rank-plot showing gene enrichment scores (normZ) of CRISPR screen hits upon camptothecin treatment in human U2OS cells. Negative scores represent dropouts, genes whose loss is predicted to promote drug sensitivity. Blue dots indicate well-characterized factors affecting cellular sensitivity to camptothecin; the red dot indicates USP37. b–e Clonogenic survival assays of control (CTRL) and USP37 knockout (KO; results from two independent clones plotted) RPE-1 TP53 KO cells upon treatment with (b) camptothecin, (c) etoposide, (d) ICRF-193, or (e) aphidicolin. n = 4 independent experiments (b–d), n = 3 independent experiments (e). Statistical analysis for (b–e) was performed using two-way ANOVA and Tukey test for multiple comparisons. f Representative images of RPA (green) and γH2AX (orange) staining in S-phase (magenta) positive cells. Scale bar: 20 μm. g, h Quantification of RPA (g) or γH2AX (h) foci in S-phase cells (EdU positive). Cells were treated with the indicated doses of the drugs for 4 h. For (g), untreated: n = 2427 (WT), 1599 (USP37 KO6), 1212 (USP37 KO17) cells; camptothecin: n = 2316 (WT), 1665 (USP37 KO6), 1350 (USP37 KO17) cells; aphidicolin: n = 2538 (WT), 1234 (USP37 KO6), 1164 (USP37 KO17) cells. For (h), same as for (g), but n = 2426 (WT, untreated), 1598 (USP37 KO6, untreated) cells. Cells were analyzed from five biological replicates, except for USP37 KO6 (four biological replicates). Bars represent the median. Black dots indicate means in each independent experiment. Statistics indicate two-tailed Wilcoxon rank-sum tests for p values without adjustment for multiple hypothesis testing and Cohen’s d for effect sizes (γ) (see methods). CPT camptothecin. i–k Clonogenic survival assays of control (CTRL) or USP37 KO (clone 10) cells expressing mCherry (EV), mCherry-USP37WT or mCherry-USP37C350A (catalytic inactive) upon treatment with camptothecin (i), etoposide (j), or aphidicolin (k); n = 4 independent experiments (i); n = 5 independent experiments (j), with the exception of “C350A” condition, where n = 4); n = 3 independent experiments (k). Bars represent means ± SEM. Statistical analysis for (i–k) was performed using two-way ANOVA and Šídák test for multiple comparisons. Source data are provided as a Source Data file.
