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. 2020 Mar 13;26:e918751-1–e918751-8. doi: 10.12659/MSM.918751

Figure 3.

Figure 3

Exo1 interacts with NHEJ required factor Ku70 and affects NHEJ efficiency. (A) Exo1 and Ku70 interact in vitro. GST-Exo1 or GST was used to capture his-Ku70 on glutathione resin. * Positive control for western blot: 3 ng of purified recombinant his-Ku70. The samples were separated using the SDS-PAGE and the his-Ku70 was examined using anti-Ku70 antibodies. (B) Schematic diagram of NHEJ reporter assay Pem1GFP. GFP coding gene is disrupted by polyadenylation sites (Ad). Ad is flanked by I-SceI sites. DSBs were introduced by transfecting of I-SceI endonuclease. After NHEJ join cohesive DNA ends and delete the Ad site from the reporter to restore GFP expression. (C) Exo1 knockout cell line showed decrease NHEJ efficiency. NHEJ efficiency of SKOV3 WT cell line was normalized to 100%. Three clones of Exo1-KO cell lines showed 45%, 55.33%, and 43.67% NHEJ efficiency. Statistically significant differences (P<0.0001) in NHEJ efficiency were determined.