Extended Data Fig. 6: Inhibition of the USP1:WDR48 DUB complex rescues cisplatin-induced SLFN11-dependent cell death in PRIMPOL KO cells.
From: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress
(a) An experimental schematic describing how cell viability assays were performed in the presence of cisplatin and ML323 (USP1 inhibitor). (b) A cisplatin dose response curve for eHAP iCas9 WT or PRIMPOL KO cells with or without 4.5 μM ML323. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. (c) A bar plot showing how eHAP iCas9 WT (gray bars) or PRIMPOL KO cells (red bars) respond to 4.5 μM ML323. Survival is normalised to untreated conditions within each genotype. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: 0.4946 (WT vs. WT + USP1i); 0.7018 (PRIMPOL KO vs. PRIMPOL KO + USP1i). (d) A schematic showing how cell viability experiments were performed in A673 Cas9-AAVS1 or A673 Cas9-PRIMPOL cells in the presence of cisplatin and ML323 (16.5 μM). (e) A bar blot depicting cell survival for A673 Cas9-AAVS1 or A673 Cas9-PRIMPOL cells in the presence of 200 nM cisplatin -/+ 16.5 μM ML323. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: 0.9925 (WT vs. WT + USP1i); 0.0203 (PRIMPOL KO vs. PRIMPOL KO + USP1i). (f) An experimental scheme depicting how cell viability was measured after challenging cells with cisplatin and USP1 inhibitor (ML323) in eHAP iCas9 cells with indicated genotypes. (g) A bar plot depicting cell survival of eHAP iCas9 WT or PRIMPOL KO cells following challenge with 670 nM cisplatin in the absence (darker bars) or presence (light bars) of 4.5 μM USP1 inhibitor following transient knockout of SLFN11. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: >0.9999 (WT sgNTC vs. WT sgNTC + USP1i); >0.9999 (WT sgSLFN11 vs. WT sgSLFN11 + USP1i); 0.0032 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgNTC + USP1i); 0.8134 (PRIMPOL KO sgSLFN11 vs. PRIMPOL KO sgSLFN11 + USP1i); 0.8522 (WT sgNTC vs. WT sgSLFN11); <0.0001 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11); 0.8160 (WT sgNTC + USP1i vs. PRIMPOL KO sgNTC + USP1i). Source numerical data are available in the source data.
