Fig. 2: CUL4A inhibits DNA DSB repair through suppression of NHEJ activity by affecting DNA-PKcs.

(A) Diagram of the working principle of the NHEJ reporter gene. (B) Schematic diagram of establishment of stable NHEJ reporter gene expression in MCF-10A cells. (C) With the NHEJ reporter gene system, I-SceI was used to induce DSBs, and flow cytometric analysis was used to analyze the effect of CUL4A on NHEJ efficiency. (D) Western blot analysis of DNA-PK kinase (DNA-PKcs, KU70/80) expression in HPDE6-C7 and CCC-HPE cells overexpressing CUL4A after DSBs induction with bleomycin. (E) DNA-PK protein kinase (DNA-PKcs, KU70/80) levels in MEFs from Cul4a transgenic mice after bleomycin treatment. (F) Expression levels of DNA-PK protein kinases (DNA-PKcs, KU70/80) in HPDE6-C7 and CCC-HPE cells with CUL4A silencing after bleomycin treatment. (G) Bleomycin induces DSBs, and the neutral comet assay results showed the accumulation of DSBs in HPDE6-C7 cells overexpressing CUL4A. (H) After treatment with bleomycin, a neutral comet assay was performed to analyze the accumulation of DSBs in MEFs from Cul4a transgenic mice. * p < 0.05, # p < 0.05, and ns = no significance based on Student’s t-test. The data are presented as the means ± SDs of five (C) or three (D–H) independent experiments. More than 100 cells were counted in G and H. The scale bars indicate 50 μm in G and H.