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. 2020 May 26;11:2641. doi: 10.1038/s41467-020-16393-4

Fig. 3. CX-5461 is synthetic lethal with HRD in HGSOC.

Fig. 3

a In vitro CX-5461 dose response proliferation time course, assessed by cell confluency using IncuCyte ZOOM of OVCAR8 and OVCAR8 RAD51C KO cell lines. Representative of two biologically independent experiments. Error bars represent mean ± SEM of n = 5 technical replicates. b Cell cycle analysis of cells treated with vehicle, 100 nM or 1 μM CX-5461 for 48 hours and 72 h and labelled with BrdU for 30 min prior to harvest. Analytical FACS analysis of BrdU incorporation as a function of DNA content was performed (Representative plots and gating strategy are shown in Supplementary Fig. 3D). Histogram plots displaying the percentage of G0/G1 (blue) and G2M (green) and S-phase BrdU-labelled (red) cell populations. Error bars represent mean ± SEM of n = 3 biologically independent experiments. c Quantitation of cell cycle profiles using FUCCI-labelled cells treated with vehicle, 100 nM or 1 μM CX-5461 for 48 and 72 h. Representative flow cytometry profiles and gating strategy are shown in Supplementary Fig. 3E. Error bars represent mean ± SEM of n = 3 biologically independent experiments. d Histogram plots displaying the percentage of cells with >4n DNA content (top panel) and Sub G0/G1 cell populations (bottom panel) as detected by BrdU/PI cell cycle analysis described in (b) and Supplementary Fig. 3D, (n = 3 biologically independent experiments, n = 7 for OVCAR8 and OVCA8 RAD51C KO cells treated with vehicle or 1 μM CX-5461 for 72 h), error bars represent mean ± SEM. Statistical analysis in B-D was performed using a two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p-values are shown). e Western blot analysis of cells treated with either vehicle, 100 nM or 1 μM CX-5461 for 6 and 24 h. Representative of n = 3 biologically independent experiments. Blots shown are of samples derived from the same experiment and were processed in parallel. Loading controls Vinculin and Actin were processed by re-probing the blots. Full sized scan of western blots are provided in Supplementary Fig. 10.