Extended Data Fig. 8: Upregulation of the Fanconi Anaemia pathway compensates for loss of PRIMPOL/RAD18 in response to cisplatin lesions.
From: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress
(a) A schematic showing how RAD18 or FANCL was knocked out in eHAP iCas9 WT or PRIMPOL KO cells and subsequently challenged with cisplatin. (b) A Western blot showing PRIMPOL and RAD18 protein levels in eHAP iCas9 WT or PRIMPOL KO cells upon loss of RAD18 (R18). (c) A cisplatin dose response curve for eHAP iCas9 WT or PRIMPOL cells upon loss of RAD18. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. These experiments were performed using the same WT sgNTC and PRIMPOL KO sgNTC samples depicted in (m). (d) A Western blot showing PRIMPOL and FANCL protein levels in eHAP iCas9 WT or PRIMPOL KO cells upon loss of FANCL (FL). (e) A cisplatin dose response curve for eHAP iCas9 WT or PRIMPOL KO cells upon loss of FANCL. eHAP iCas9 WT and PRIMPOL KO curves are the same as those shown in (d), as these experiments were performed at the same time. These experiments were performed in N = 3 biological replicates. Data are represented as means ± SD. (f) A series of Western blots showing indicated protein levels in eHAP iCas9 WT or PRIMPOL cells upon loss of USP1 and/or RAD18 (R18). (g) A series of Western blots showing indicated protein levels in eHAP iCas9 WT or PRIMPOL KO cells upon loss of USP1 and/or FANCL (FL). (h) An experimental scheme for how USP1/RAD18/FANCL proteins were knocked out in eHAP iCas9 WT or PRIMPOL KO cells and subsequently challenged with cisplatin in a colony formation assay. (i) Representative images of colony formation assays in indicated cell backgrounds in untreated conditions or upon treatment with 450 nM cisplatin. (j) Quantification of colony survival in the colony formation assays represented in (d) normalised to untreated samples 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 test statistical significance. P-values: 0.9964 (WT sgNTC vs. WT sgUSP1); 0.0098 (WT sgRAD18/NTC vs. WT sgRAD18/sgUSP1); 0.9998 (WT sgFANCL/sgNTC vs. WT sgFANCL/USP1); 0.0012 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgUSP1); <0.0001 (PRIMPOL KO sgRAD18/NTC vs. PRIMPOL KO sgRAD18/USP1); >0.9999 (PRIMPOL KO sgFANCL/NTC vs. PRIMPOL KO sgFANCL/USP1). (k) An experimental schematic describing how chromatin-bound immunofluorescence was performed. (l) Representative micrographs depicting DNA staining (DAPI), FANCD2 staining, and a merged image of DAPI/FANCD2 staining. (m) A bar plot showing chromatin-bound FANCD2 foci numbers in eHAP iCas9 WT or PRIMPOL KO cells treated with 450 nM cisplatin. These experiments were performed in N = 5 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess statistical significance. P-values: 0.0071 (WT + cisplatin vs. PRIMPOL KO + cisplatin). Source numerical data and unprocessed blots are available in the source data.
