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

Fig. 6. CX-5461-induces replication-dependent DNA damage in HR-proficient HGSOC cells.

Fig. 6

a Co-IF analysis of γH2AX in cells labelled with EdU and treated with vehicle or 1 μM CX-5461 for 3 hours. Representative images of three biologically independent experiments. b Quantitation of foci count was performed using CellProfiler. n = 250 EdU positive cells and n = 220 EdU negative cells per treatment condition were examined over three independent experiments. Error bars represent mean ± SD. c IdU track length is reduced by CX-5461 through MRE11-dependent mechanism. Schematic of CIdU and IdU pulse-labelling method used (top). OVCAR8 cells were sequentially labelled and either processed or treated with 1 μM CX-5461, 50 mM mirin or the combination of both for 3 h. Fibres were processed for DNA fibre analysis. Replication Fork lengths were calculated based on the length of individual IdU tracks measured using ImageJ software. IdU track lengths (in μm) were converted to kb (1 kb = 2.59 μm). n = 150 replication tracks were analysed over two biologically independent experiments. Error bars represent mean ± SD. d DNA fibre analysis of OVCAR8 cells pre-treated with 100 nM CX-5461, 100 nM BMN-673 or in combination, washed then sequentially labelled with CldU and IdU as indicated in the schematic (top). Fibres were processed and analyzed as described above. n = 150 replication tracks were analysed over two independent experiments. Error bars represent mean ± SD. Statistical analysis (in b–d) was performed using a two-sided one-way ANOVA, Kruskal–wallis multiple comparisons test (adjusted p-values are shown). NS denotes a non-significant p-value > 0.05.