FIGURE 7.

Kdm4b overexpression promotes DSB repair. A, U2OS cells were plated on glass coverslips and transfected with Kdm4b-EGFP. 24 h after transfection, the plated cells were treated with 2 Gy of γ-irradiation (γIR) and fixed at 1, 6, and 24 h post-irradiation. Cells were stained by immunofluorescence for γH2AX, and the nuclei were counterstained with DAPI. The numbers of γH2AX foci were counted after 1, 6, and 24 h recovery in both Kdm4b-EGFP-positive cells and neighboring untransfected (control) cells. The mean number of foci is indicated by the horizontal bar. Statistical differences were determined by using a two-tailed t test. n.s., not significant. B, levels of Kdm4b-EGFP were negatively correlated with the number of remaining γH2AX foci after 6 h recovery. Correlation was measured using one-tailed Pearson correlation calculation. The mean EGFP intensity was measured for individual masked nuclei using CellProfiler. C, two examples show the inverse correlation between Kdm4b-EGFP levels and γH2AX foci at 6 h post-irradiation. D, U2OS cells were plated onto glass coverslips and transfected with either wild type Kdm4b-EGFP or inactive Kdm4b^H189A-EGFP. At 24 and 48 h post-transfection, coverslips were fixed and stained for γH2AX foci, and the nuclei were counterstained with DAPI. Foci numbers were counted for the transfected cells as well as the EGFP-negative control cells. Kdm4^H189A-EGFP/48h had a significantly (p < 0.001) higher number of γH2AX foci than any other experimental condition (one way analysis of variance); all other comparisons did not show significant differences.