Fig. 3

ID3 interacts with MDC1 to promote DDR in colorectal cancer. A Western blotting (left panel) and statistical analysis (right panel) of ID3, MDC1 and γH2AX protein expression after irradiation. The protein expression of ID3, MDC1 and γH2AX all increased significantly after irradiation. B Immunofluorescence revealed ID3, MDC1, and γH2AX foci formation after irradiation of HCT116 cells. C Comet tail moments (left panel) and statistical analysis (right panel) at indicated time points after exposure to IR in HCT116 cells. D Comet tail moments (left panel) and statistical analysis (right panel) in both control and ID3-depleted colon cancer cells after exposure to IR. E Western blotting (left panel) and statistical analysis (right panel) of ID3, MDC1, γH2AX and PPARγ expression with ID3 knockdown. There was no significant change in γH2AX expression when ID3 expression decreased, but the expression of MDC1 and PPARγ decreased significantly. F Lysates of HCT116 cells were subjected to co-immunoprecipitation using an anti-Myc antibody followed by western blotting (left panel) using anti-MDC1 and anti-ID3 antibodies. A statistical graph is located on its right. The result showed that MDC1 interacted with ID3. G Immunofluorescence showed ID3 and MDC1 colocalization in HCT116 cells with or without exposure to X-ray irradiation. The cells were exposed to 6 Gy irradiation and fixed at the indicated time points. β-actin was used as a loading control. Experiments were repeated at least three times. Data are expressed as mean ± SD (n = 3). *P < 0.05. NC: siRNA control, PC: pcDNA3.1 control, KD: knockdown, OE: overexpress, Ctrl: radiation control