Fig. 2: WDR48–USP1 counteracts RAD18-driven PCNA degradation.
From: Comprehensive interrogation of synthetic lethality in the DNA damage response
a, Dual-colour flow cytometry assay of the LIG1:WDR48 interaction in cells expressing a control (CTRL) or RAD18-targeting sgRNA. Lines represent mean (three technical replicates). b, Similar to a, but in this case, cells were complemented with the indicated doxycycline (DOX)-inducible cDNAs. Day 10 values are shown. Error bars represent mean ± s.d. (three technical replicates). c, Western blot analysis of the indicated proteins. Repeated once with similar results. d, Top, schematic of the PIP–FUCCI reporter, experimental set-up and representative images. USP1i, 20 μM. Bottom, quantification of S phase length (two experimental replicates). At least 40 cells were measured per condition. The orange line denotes the median. Statistical analysis was performed using Kruskal–Wallis ANOVA. e, Left, representative flow cytometry plots of EdU incorporation and DNA content. Cells were treated with USP1i (25 μM) for 2 days. Samples were treated with 1 μg ml−1 DOX. Right, quantification of EdU incorporation in G2/M phase cells (three experimental replicates). Error bars represent mean ± s.d.; unpaired two-tailed Student’s t-test. f, Left, representative image of a UFB. Right, corresponding quantification (two experimental replicates). Cells stably expressing H2B–GFP were treated with USP1i (25 μM) for 24 h then imaged for an extra 24 h. Error bars represent mean ± s.d.; unpaired two-tailed Student’s t-test. g, Left, representative images of cyclin A and 53BP1 staining. Cells were treated with USP1i (25 μM) for 48 h. Right, corresponding quantification. Box plots show median values and data within the 10–90 percentile. The lower and upper ends indicate the 25th and 75th percentiles. At least 75 cells from two experimental replicates were quantified. Statistical analysis was performed using Kruskal–Wallis ANOVA. h, A model for the FEN1/LIG1:WDR48–USP1 synthetic lethal interactions. In d–g, USP1i refers to KSQ-4279. EV, empty vector; LE, long exposure; SE, short exposure; Ub, ubiquitylated; WT, wild type. Scale bars, 10 μm (d,g), 25 μm (f).
