Fig. 2: Origin firing in POLE1-depleted cells.

a, b Wild-type U2OS or homozygous mAID-KI clone 16 were treated for 16 h with DMSO or dox/aux, 5 µM ATRi was added to the indicated samples for 1 h, followed by cell lysis and the isolation of the insoluble chromatin fraction. Equal amounts of protein were loaded. Western blot of the soluble lysates (a) and insoluble chromatin fraction (b) is shown. 1 h hydroxyurea (HU, 2 mM) treatment was used as a positive control for replication stress. The second band on POLE1 blot (b) likely represents partially degraded protein. Specific signals of SLD5 and CDC45 were quantified by Fiji/ImageJ. c Indicated cell lines were synchronized by thymidine/nocodazole blocks and treated with dox/aux as indicated. Western blot analysis of chromatin from the cells collected at the indicated timepoints is shown. Equal amounts of protein were loaded. Specific signals of SLD5 and CDC45 were quantified by Fiji/ImageJ. d, e Clone 16 cells were treated for 16 h with DMSO or dox/aux as indicated, DMSO or 5 µM ATRi was added to the indicated samples for 60 min before harvest, 10 µM EdU was added for the last 30 min of treatment. Flow cytometry plots showing EdU incorporation histograms (c) and relative EdU incorporation, normalized to the samples without ATRi, are shown (d)—mean + SD from n = 3 (DMSO) or n = 4 (dox/aux) independent experiments. Paired t-test was used for statistical analyses, p values are shown. f Clone 16 cells were incubated with 10 µM CldU for 48 h, DMSO or dox/aux were added for the last 16 h of treatment. After CSK extraction, cells were fixed and stained with anti-CldU antibodies under native conditions. Quantification of ssDNA-positive cells is shown—mean + SD from n = 3 independent experiments. Paired t-test was used for statistical analyses, p value is shown. Source data are provided as a Source data file.