Figure 1.
NRAGE is required for efficient homologous recombination repair (HRR) in esophageal cancer cells. (a) Total protein from EC9706, EC109, and TE1, stably knockdown of NRAGE using lentivirus mediated shRNA plasmids, were subjected to western blotting assays with the indicated antibodies. (b) The γH2AX foci formed in EC9706/lv-shCon. and EC9706/lv-shNRG cells were detected using IF, the scale bar is 5 and 2 μm, respectively. Cells with more than three γH2AX foci in the nucleus were defined as positive cells. The γH2AX foci-positive cells out of 300 cells (γH2AX foci positive%) were represented as means±S.D. and statistically analyzed (*P=0.037). (c) EC109/lv-shCon. and EC109/lv-shNRG cells were subjected to neutral comet assays to evaluate DSBs, the scale bar is 100 μm. OTM of 300 cells were statistically analyzed (**P=0.001) and graphically depicted. Error bar represents S.D. NRAGE interference efficiency was confirmed by western blottings. (d) 293T cells transfected with siCon. or siNRG, together with DR-GFP (−), GFP (+), NHEJ, or DR-GFP+Isce-I (HR), respectively, were subjected to flow cytometry to test GFP percentage (GFP%) in about 1 × 104 cells. The HR and NHEJ efficiency of siCon. and siNRG (#1, #2) cells were normalized to corresponding GFP (+). Data from three independent experiments were represented as means±S.D. and statistically analyzed (**P=0.006 for siNRG #1 and *P=0.04 for siNRG #2 in HR efficiency analysis). The efficiency of NRAGE knockdown was confirmed by western blottings. (e) 293T cells transfected with Flag or Flag-NRG, together with DR-GFP (−), GFP (+), or DR-GFP +Isce-I (HR) or NHEJ, respectively, were subjected to flow cytometry to test GFP%. The HR or NHEJ efficiency of Flag or Flag-NRG-transfected cells were normalized to the corresponding GFP (+), respectively. Data from three independent experiments are represented as means±S.D. and statistically analyzed (**P=0.002 for HR efficiency analysis). Overexpression of NRAGE was confirmed by western blottings