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

Figure 1.

Figure 1

Inhibition of WIP1 impairs homologous recombination (HR). (A) Traffic light reporter assay in U2OS cells. Two independent stable cell lines (clones #10 and #12) were transfected with ISceI together with BFP-donor vector with or without pretreatment with 1 μM WIP1i. Efficiency of repair was analyzed 3 days after transfection by FACS. Plotted is mean of normalized ratio of GFP+/RFP+ cells. Bars indicate SD, n ≥ 3. Statistical significance evaluated by two-tailed t-test. (B) Traffic light reporter assay in two independent clones of RPE cells (#3 and #4). Same as A. (C) Efficiency of repair by HR (GFP+) and NHEJ (RFP+) in TLR assay in U2OS cells from A. (D) Efficiency of repair by HR (GFP+) and NHEJ (RFP+) in TLR assay in RPE cells from B. (E) Cell survival after irradiation of parental U2OS and two independent U2OS-WIP1-KO cell lines treated or not with WIP1 inhibitor was evaluated after 7 days using resazurin viability assay. Plotted is mean and SD, n ≥ 3. Statistical significance evaluated by two-way ANOVA (* P < 0.05; *** P < 0.001). (F) Cell survival of parental U2OS and two independent U2OS-WIP1-KO cell lines treated with indicated doses of camptothecin with or without combined treatment with WIP1 inhibitor was evaluated after 7 days using resazurin viability assay. Plotted is mean and SD, n ≥ 3. Statistical significance evaluated by two-way ANOVA (* P < 0.05; *** P < 0.001). (G) Cell survival after irradiation of parental RPE and RPE-WIP1-KO cell lines assayed as in E. (H) Cell survival of parental RPE and RPE-WIP1-KO cell lines with treated with camptothecin and analyzed as in F. (I) Percentage of dead cells was evaluated by Hoechst 33258 staining and FACS analysis 7 days after treatment with camptothecin or after irradiation in U2OS cell line with or without combined treatment with WIP1i. Plotted is mean +/− SD. Statistical significance evaluated by two-tailed t-test.