Extended Data Fig. 4: Cisplatin induces ssDNA accumulation, SLFN11 chromatin loading, induction of the ISR, and apoptosis in the absence of PRIMPOL-mediated repriming.
From: RPA exhaustion activates SLFN11 to eliminate cells with heightened replication stress
(a) Representative flow cytometry experiments depicting cleaved caspase-3 signal plotted against forward scatter area. Cleaved caspase-3 positive cells were gated and quantitated as depicted. (b) A scheme depicting how apoptosis was measured in eHAP iCas9 WT or PRIMPOL KO cells using cleaved caspase-3 positive cells in flow cyomtetry experiments. (c) Representative dot plots depicting how the percentage of cleaved caspase-3 positive cells were determined using flow cytometry after challenge with cisplatin in the absence or presence of 650 nM GCN2 inhibitor. (d) A bar plot depicting the percentage of cleaved caspase-3 positive cells in each indicated genotype following challenge with cisplatin in the absence or presence of GCN2 inhibitor determined using flow cytometry in (b). These experiments were performed in N = 4 biological replicates. Data represented as means ± SD. A two-way ANOVA was performed to assess biological significance P-values: 0.0003 (WT sgNTC vs. PRIMPOL KO sgNTC + cisplatin); >0.9999 (WT sgSLFN11 vs. PRIMPOL KO sgSLFN11 + cisplatin); 0.0002 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin). (e) Representative Western blots demonstrating induction of DNA damage response markers in eHAP iCas9 WT or PRIMPOL KO cells in response to indicated doses of cisplatin for 24 or 48 h. (f) Experimental setup of measuring chromatin-bound phospho-gamma H2AX serine 139 using immunofluorescence in eHAP iCas9 WT or PRIMPOL KO cells depleted of SLFN11 protein (top). (g) eHAP iCas9 WT or PRIMPOL KO Cells transiently depleted of SLFN11 were mock treated or treated with 450 nM cisplatin for 24 h. Representative micrographs of chromatin-bound immunofluorescence of γH2AX phospho serine 139, DAPI, or merged are shown. (h) Bar plots showing phospho-gamma H2AX serine 139 foci signal normalised to WT untreated cells in eHAP iCas9 WT or PRIMPOL KO cells depleted of SLFN11 protein. Experiments were performed in N = 5 biological replicates. Data are represented as means ± SD. A two-way ANOVA was used to assess biological significance. P-values: >0.9999 (WT sgNTC vs. PRIMPOL KO sgNTC + cisplatin); 0.4353 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin); 0.2441 (WT sgNTC vs. PRIMPOL KO sgSLFN11). γH2AX phospho serine 139 staining was performed as a co-stain with pRPA S33 depicted in Fig. 4j-l. (i) eHAP iCas9 WT or PRIMPOL KO Cells transiently depleted of SLFN11 were mock treated or treated with 450 nM cisplatin for 24 h. Representative micrographs of chromatin-bound immunofluorescence of RAD51, DAPI, or merged are shown (j) Quantification of RAD51 foci in indicated cell lines. These experiments were performed in N = 5 biological replicates. Data are represented as means ± SD. A two-way ANOVA was performed to assess biological significance. P-values: 0.0050 (WT sgNTC vs. PRIMPOL KO sgNTC + cisplatin); 0.0013 (PRIMPOL KO sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin); 0.9994 (WT sgNTC vs. PRIMPOL KO sgSLFN11 + cisplatin). Source numerical data and unprocessed blots are available in the source data.
