FIGURE 2.

Rsf-1 induces DNA strand breaks and activates DDR. IOSE-80pc cells were used for studying DNA damage response (DDR) induced by Rsf-1 overexpression. A, Western blot analysis shows that Rsf-1 overexpression in ovarian surface epithelial cells is associated with increased protein levels of phosphorylated γ-histone 2AX (γH2AX), phosphorylated check point kinase-2 (pCHK2), p53, and p21. GAPDH serves as a loading control. B, upon gel electrophoresis, DNA with strand breaks migrated out of nuclei forming a comet tail-like structure, whereas the nondamaged DNA remained stationary within the nucleus. At all time points, the percentage of comet-like cells was significantly higher in the Rsf-1-overexpressing group than in the control group (p < 0.001). UV-irradiated cells serve as the positive control. C, immunofluorescence staining demonstrates punctuate immunofluorescence (foci) of both γH2AX and pCHK2 in cells at 36 and 48 h after Rsf-1 transduction. The percentages of Rsf-1, γH2AX, and pCHK2-positive cells at different time points were counted and shown in the lower panel. Error bar: one standard error.