Fig. 7.

TR3 represses DSB repair in response to IR. A, The overexpression of TR3 results in the suppression of DSB repair. HepG2 cells (left) and HA-TR3-overexpressing HepG2 cells (right) were exposed to IR (3 Gy). The cells were then harvested at various time points as indicated. DSB repair was determined by analyzing γ-H2AX via immunostaining with a specific primary γ-H2AX antibody followed by a fluorescein isothiocyanate-conjugated secondary antibody. HA-TR3 proteins were detected by incubation with an anti-HA antibody followed by a Texas-Red-conjugated secondary antibody. Stained cells were visualized using confocal microscopy. B, The number of γ-H2AX foci per cell was determined on a cell-to-cell basis by quantitative analysis of the above samples. C, Effects of TR3 on the IR-induced expression of γ-H2AX. HepG2 cells and Myc-TR3 expressing HepG2 cells were exposed to IR (3 Gy). After 24 h, the cell lysate was analyzed via Western blotting using anti-γ-H2AX antibodies. The relative expression levels for γ-H2AX were quantified using densitometry. The γ-H2AX levels at 1 h after IR exposure in each panel were normalized to 1. D, TR3-enhanced γ-H2AX expression depends on the presence of Ku80. HepG2 cells were transfected with si-Ku80 and TR3 as indicated, and the scrambled siRNA was used as a control (si-Ctrl). The methods of detection and quantification were the same as those described above. E, TR3 inhibits Ku80 and DNA-PK binding to DNA upon IR. HepG2 cells were transfected with HA-TR3. The nuclear extracts were incubated with either 18-bp biotinylated double-stranded oligonucleotides for Ku80 and DNA-PKcs or streptavidin-agarose beads, but no probe, as a control and then analyzed via DAPA. Specifically bound proteins were analyzed via Western blot analysis using antibodies specific for DNA-PKcs and Ku80, and the expression level of PARP was assessed as a loading control. DAPI, 4′,6-Diamidino-2-phenylindole.