Figure 3. CX-5461 and CX-3543 induced DNA damage is replication-dependent.

(a) Active replication decreased upon CX-5461 treatment in WT and BRCA2^−/− HCT116. Cells were treated with CX-5461 for the time indicated before incubating with EdU (10 μM) for 1 h. Cells were analysed by FACS with the intensity of EdU and PI recorded. Left panel shows one representative FACS profile when cells were treated with CX-5461 at 10⁻⁶ M; right panel shows the mean percentage of cells in S phase (with 95% CIs) under different CX-5461 concentrations at different time points; n=3 experiments. Cell cycle distributions at more time points and drug concentrations are shown in Supplementary Fig. 5a and Supplementary Table 6. (b) CX-5461 induced 53BP1 foci enriched in S phase (positive for EdU labelling), and APH greatly suppressed CX-5461 induced DNA damage in HCT116. WT Cells were treated with EdU (20 μM) for 30 min, then EdU was washed out and the cells were treated with CX-5461 (10⁻⁷ M) for 1 h. For APH treatment, after EdU labelling, APH (5 μM) was added for 2 h before incubating with CX-5461 (10⁻⁷ M) for 1 h. Scale bar, 10 μM. (c) The percentage of 53BP1 foci positive cells within EdU positive and EdU negative population with or without APH was quantified in HCT116 cells. Experimental conditions were the same as stated in b. Bars show the mean of three time course experiments (>100 cells each replica) and 95% CIs. (d) Replication rate is reduced by CX-5461 in BRCA2 deficient cells at higher level than in BRCA2 proficient cells. CIdU (30 min) treated HCT116 cells were chased with or without CX-5461 for 30 min in the presence of IdU, then the cells were processed for DNA fibre analysis; n=2. Median fork rate and the number of tracks analysed are shown. The box extends from the 25th to 75th percentiles. P value was calculated by Mann–Whitney U test.