Fig. 2

PARGi sensitivity is accompanied by markers of replication stress and the DNA damage response. A Representative images of RPA1 and γH2AX foci in response to 48 h of 1 μM PARGi treatment or DMSO (Control) in PARGi-resistant (COV318) and PARGi-sensitive (OVMANA) cell lines (upper). RPA1 and pan-nuclear γH2AX staining also shown in PARGi-sensitive OVISE cells following 72 h PARGi treatment (lower). Scale bars: 20 μm. B Representative images of pan-nuclear pKAP1 and γH2AX staining in PARGi-resistant (COV362) and PARGi-sensitive (OVISE) cell lines following 72 h of 1 μM PARGi treatment. Scale bar: 20 μm. C Representative immunoblot Li-COR image for PARGi-resistant (OVCAR3) and PARGi-sensitive (Kuramochi) cell lines; cells were treated for 48 h with 1 μM PARGi (Gi) or DMSO as a negative control (C), or for 2 h with 2 mM hydroxyurea (H) as a positive control. D Bubble plot showing fold-change in RPA1 foci, γH2AX foci, pan-nuclear pKAP1 and Chk1 phosphorylation in response to PARGi treatment. Mean of n ≥ 3 biological replicates for each parameter quantified using single-cell immunofluorescence microscopy. Bubble size represents pChk1, bubble colour represents pKAP1. (E) Schematic of DNA fibre experimental strategy. (F) Exemplar fibres in PARGi-resistant (COV318) and PARGi-sensitive (OVMANA) cell lines. Scale bar: 10 μm. (G) Mean % asymmetric forks are shown on the left. The exemplar graphs on the right show correlation between R and L fork lengths. Dotted lines represent 33% cut-off, beyond which forks are considered asymmetric (% asymmetry for replicate shown in respective graph bottom right corner). Statistics: Mean of 3 biological replicates, with ≥50 forks measured per cell line, per condition. 2-way ANOVA with Sidak post-hoc test, selected comparisons were between PARGi treated values and DMSO control within each cell line. Error bars represent SEM. * p < 0.05. See also Fig. S3