Fig. 4: Drug effects on homologous recombination and DNA double-strand breaks. | Nature Communications

Fig. 4: Drug effects on homologous recombination and DNA double-strand breaks.

From: Combining PARP with ATR inhibition overcomes PARP inhibitor and platinum resistance in ovarian cancer models

Fig. 4

a–c Parental BRCAMUT (BRCA2MUT PEO1; BRCA1MUT JHOS4, UWB), PARPi-resistant (PEO1-PR, JHOS4-PR, Kuramochi, UWB/B1 where B1 denotes BRCA1+/−), and platinum-resistant (PEO1-CR, PEO4; CCNE1Amp OVCAR3, FUOV1) were treated with PARPi (1 μM), ATRi (1 μM), or combination for 24 h. a, b RAD51 (green) nuclear foci were detected in geminin positive (red) cells by immunofluorescence staining. Magnification is ×60 for large panel and ×100 for insert. Scale bar = 20 μm. b Cells with >5 foci in the nucleus were counted as a positive RAD51 cell. Each dot represents the mean number of events per high-power fields (n = 5 fields) with mean ± SD shown. RAD51 in PARPi group was higher than control for PEO1-PR (P < 0.0001), UWB/B1 (P < 0.0001), PEO4 (P < 0.0001), FUOV1 (P < 0.0001), PEO1-CR (P = 0.002), and OVCAR3 (P = 0.0003) lines, and RAD51 in PARPi–ATRi treatment group was lower than PARPi monotherapy for these cell lines (P < 0.0001). c Detection of \({\upgamma}\)H2AX-positive cells in S phase of PARPi–ATRi-treated parental BRCAMUT, PARPi-resistant, and platinum-resistant cells. Cells were treated with 1 μM PARPi, 0.5 μM ATRi or combination, and JHOS4-PR and OVCAR3 were treated with 1 μM PARPi, 1 μM ATRi, or combination. After treatment for 24 h (JHOS4-PR 36 h), cells were fixed and stained with \({\upgamma}\)H2AX and PI for flow cytometry, and \({\upgamma}\)H2AX-positive cells in S phase were quantified. Representative images of PEO1-PR cells (left) and quantified data (right) were shown. \({\upgamma}\)H2AX-positive cells in PARPi–ATRi group was higher than ATRi monotherapy for all cell lines (JHOS4, P = 0.0003; FUOV1, P = 0.0002; and remaining cells, P < 0.0001) except OVKATE, P = 0.3703). Data shown is mean ± SD (n = 3 biologically independent samples). Individual samples are presented as data points. Data was analyzed by one-way ANOVA test followed by Tukey’s multiple-comparisons test; ***P < 0.001, NS = not significant. Source data are provided as a source data file.

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