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. 2019 Oct 15;8(10):1258. doi: 10.3390/cells8101258

Figure 2.

Figure 2

WIP1 plays role in DNA double-strand break repair in S-phase cells. (A) Quantification of 53BP1 foci in replicating (EdU+) cells after irradiation. U2OS parental cell lines with or without combined treatment with WIP1i and two independent WIP1 knockout cell lines were pulse-labeled with EdU for 30 min before irradiation. Cells were fixed after pre-extraction at indicated time-points and stained with 53BP1 antibody. Click chemistry was used to visualize EdU. Mean of median foci number +/- SD is plotted (n ≥ 3). Statistical significance evaluated by two tailed t-test. (B) Quantification of 53BP1 foci in non-replicating (EdU-) cells after irradiation. As in A. (C) Quantification of 53BP1 foci in replicating (EdU+) cells after irradiation. U2OS parental, WIP1 knockout and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 were irradiated and analyzed as in A. (D) Quantification of 53BP1 foci in non-replicating (EdU-) cells after irradiation. U2OS parental, WIP1 knockout and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 were irradiated and analyzed as in A. (E) Traffic light reporter assay in U2OS cells after transfection with indicated siRNA. Cells were transfected with ISceI together with BFP-donor vector with or without pretreatment with 1 μM WIP1i 2 days after siRNA transfection. Efficiency of repair was analyzed by FACS 3 days after ISceI and BFP-donor transfection. Plotted is mean +/− SD. Statistical significance evaluated by two-tailed t-test. (F) Efficiency of repair by HR and NHEJ in Traffic light reporter assay as in E. (G) Representative plots from Traffic light reporter assay in E.