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. 2022 Oct 14;36(11):2605–2620. doi: 10.1038/s41375-022-01708-9

Fig. 4. DDX41 inhibition induces mild DNA replication stress in S phase.

Fig. 4

A Suppression of HeLa and K562 cell proliferation by DDX41 inhibition (50 μM DDX41inh-2 treatment). Cell number was counted by using trypan blue. Values are means ± SD of triplicate samples; two-tailed unpaired Student’s t test. B Reduced BrdU incorporation by 50 μM DDX41inh-2 treatment in HeLa cells. C Schematic diagram of cell cycle synchronization, drug treatment, and IdU incorporation in HeLa cells. D Reduced IdU incorporation in S phase by 50 μM DDX41inh-2 treatment in HeLa cells. *p < 0.0001, two-tailed unpaired Student’s t test. Results of the 8-h treatment are not shown because S-G2 transition occurred (see Supplementary Fig. S4B). E Delayed S-phase progression in HeLa cells by 50 μM DDX41inh-2 treatment. (Left) Representative histograms. (Right) DNA synthesis rate as estimated by median fluorescence intensity (MFI) of PI. Values are means ± SD of triplicate samples; *p < 0.0001; †p < 0.005, two-tailed unpaired Student’s t test. F Slowed replication fork progression by DDX41 inhibition. (Left) Experimental scheme of dual labeling with DNA analogs and representative images of DNA fibers. Thymidine analogues were visualized via immunofluorescence (CldU, green; IdU, red). (Right) Fork progression speed was calculated for each sample. Bars represent means ± SD; n = 139 and 134 for DMSO and DDX41inh-2 group, respectively; two-tailed Welch’s t test. G Increase in single-stranded DNA by treatment with 50 μM DDX41inh-2 or 10 nM APH. Scale bars: 10 μm. Bars represent means ± SD; n = 400, 380 and 222 nuclei for DMSO, DDX41inh-2, and APH, respectively; two-tailed Welch’s t test. H Increase in DNA damage-related signals by DDX41 inhibition in HeLa cells. Protein extracts obtained from cell cycle-synchronized HeLa cells were probed with antibodies indicated at left. The time indicated are the hours after release from G1/S arrest. I Schematic diagram of mitosis assessment in HeLa cells after 50 μM DDX41inh-2 treatment during S phase. RO-3306, a CDK1 inhibitor, was used to induce G2 arrest. J, K Delayed mitosis in HeLa cells after DDX41 inhibition in S phase. HeLa cells were treated as indicated in I. Flow cytometry analysis of cell cycle change (J) and proportion of cells at M phase (K). Cells at G2 or M were determined as those negative or positive for pHH3 with PI signal corresponding to 4N, respectively. Right panel in K indicates proportion of pHH3-positive M phase cells 1 h after RO-3306 removal. Bars indicate means, error bars indicate SD of triplicate samples; two-tailed Welch’s t test.