Extended Data Fig. 9: Control of replication origin by MRD factor WEE1 kinase.

a, Dot plot depicting the Z-score based on EdU intensity ratios using a kinome library siRNA screen. The screening strategy is like the one described in Extended Data Fig. 6a. Top three hits are marked. The Z-score rank is based on two independent repeats. CHEK1 was not selected for downstream assays due to the pan-nuclear staining pattern of γH2AX after knockdown (data not shown). b, (top) Western blot for PKMYT1 and WEE1 proteins after 72 h of siRNA knockdown in U2OS cells. Different sets of siRNAs are used compared to the primary screen. H3 is used as a loading control. (bottom) Violin plots showing EdU ratio (A/B) elucidated from SR microscopy after siRNA knockdown. Mean EdU intensity ratios are shown on top of each violin. A minimum of 100 cells were analyzed. Representative of two biological replicates. c, Representative EdU cell cycle plots of WEE1i-treated U2OS cells. DMSO-treated cells are used as control. Hyper-replication fraction is marked using a dotted line with the percentage and standard deviation. The numbers are from three independent experiments. d,e, DNA combing experiments in DMSO or WEE1i-treated (1 h) U2OS cells. d, Representative images of DNA fibers labeled with DNA (blue), IdU (green), and CldU (red) taken from DMSO and WEE1i-treated cells. IdU treatment is initiated at 1 h post-DMSO or WEE1i treatment. A minimum of 78 fibers are analyzed in each condition. Scale bar is 50 microns. e, Graphs showing quantitative analyses of the DNA combing assay comparing DMSO or WEE1i-treated U2OS cells. Replication fork speed (left), inter-origin distance (center) and number of origins per field (right) are shown. p-values from two-sided Wilcoxon signed rank. p = 2.2e-16 was displayed when p-value was equal or smaller than 2.2e-16. f, Violin plots depicting log₂ fold change in BrdU after WEE1i treatment across the genome classified according to replication timing. Control AsiSI-U2OS cells are shown. Replication timing regions are taken from Extended Data Fig. 2b. Median values are provided on top of the violins. All p-values from two-sided Wilcoxon signed rank test are <2.2e-16. Representative of 2 independent experiments. g,h, Analysis of genomic distribution of γH2AX following WEE1i. Violin plots depicting log₂ fold change in γH2AX after WEE1i treatment across the genome, classified according to (g) replication timing domains, and (h) euchromatin (H3K4me3) vs heterochromatin (H3K27me3) domains. Histone marks are from⁷⁴. Median values are shown on top of the violins. p-values from Wilcoxon signed rank test between early vs. late and H3K4me3 v.s H3K27me3 are <2.2e-16. Results are averaged from 2 independent experiments. i, γH2AX and EdU cell cycle flow cytometry plots of U2OS cells treated with DMSO or WEE1i for 1 h. γH2AX-positive fraction is gated and is shown in red color with the average percentage and standard deviation. Hyper-replication fraction is shown using a dotted line. This plot is representative of three independent experiments.