Fig. 4: CXorf56 protein inhibits NHEJ and promotes HR, increasing resistance to DNA-damaging agents.

A Confirmation of CXorf56 protein knockdown (shCX#1 and shCX#2) and re-expression (rescued#1 and rescued#2) in MDA-MB-231 cells. The sensitivity of control (Ctrl), Cxorf56-knockdown, or CXorf56 re-expression MDA-MB-231 cells to IR (B), Cisplatin (C), and Olaparib (D) was assessed by colony formation assays. Data were analyzed by a two-tailed t test. E, F Ctrl, CXorf56-knockdown, or CXorf56 re-expression MDA-MB-231 cells, were transfected with NHEJ reporter (EJ5-GFP) or HR reporter (DR-GFP) along with pCBA-I-SceI and mCherry. Forty-eight hours later, cells were harvested and subjected to flow cytometric analysis. Data were analyzed by a two-tailed t test. G Analysis shows linear regressions and Pearson correlations between relative NHEJ and HR efficiency in Ctrl, CXorf56-knockdown, and CXorf56 re-expression MDA-MB-231 cells. Data were analyzed by a F test. H Cell cycle analyses of Ctrl, CXorf56-knockdown, or CXorf56 re-expression MDA-MB-231 cells show that the change of CXorf56 protein levels did not alter the cell cycle distribution of MDA-MB-231 cells. Data were analyzed by ANOVA and two-tailed t test. I, J Ctrl, CXorf56-knockdown, and CXorf56 re-expression MDA-MB-231 cells were treated with IR (1 Gy, 1 hour for MDC1, 53BP1, Ku70; 1 Gy, 3 hours for RPA32; 1 Gy, 5 hours for BRAC2 and RAD51), and indicated foci were detected by immunofluorescence. Representative images are shown in i. Quantification of focus signals is shown in J. Data were analyzed by ANOVA and two-tailed t test. Remarks: ^nsp ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001; Data are presented as mean ± SD. Scale bars = 10 μm.