Fig. 1.
ATR inhibition induces local dormant origin firing throughout the S phase. (A–C) U2OS or BJ-hTERT cells were treated with vehicle or 5 M AZD6738 for 45 min, and EdU was added for the last 15 min of treatment. EdU was detected by click-chemistry reaction, and PCNA was detected with fluorescently tagged antibodies. (A) Representative image of EdU staining is shown. (B and C) Fluorophore per EdU (B) or PCNA (C) focus (FPF) was quantified by STORM imaging and Auto-PC analysis of individual nucleus. One data point displays the average FPF of the foci within one nucleus. The data from three independent experiments are pulled together. t test was used for statistical analyses. ***P < 0.0005; ****P < 0.0001. (D–F) U2OS or BJ-hTERT cells were treated with vehicle or 5 M AZD6738 for 45 min, and EdU was added for the last 15 min of treatment. (D) FACS analysis of EdU/DNA content is shown. (E and F) Fold increase in EdU incorporation after ATR inhibition is quantified from three independent experimental repeats. t test was used for statistical analyses. Cells with 2N-3N DNA content were considered “early S” and 3N-4N “late S.” (G) Repli-seq correlation between log ratio of early to late at LOESS smoothing step of 50-kb windows along the genome of the samples. R, Pearson correlation coefficient. (H) Replication-timing (RT) profiles of U2OS–vehicle and U2OS–AZD6738 for the whole genome and chr7 after LOESS-smoothing step. Data were visualized by using IGV.