FIGURE 4.

Loss of RFWD3 leads to delayed recruitment of Rad51, persistent Rad51, and γH2AX foci and increased DNA damage sensitivity. A, U2OS cells transfected with RFWD3 or control siRNA were treated with 2 mm HU and fixed at the indicated time to perform Rad51 immunostaining. More than 200 cells were counted at each time point. Cell with more than 10 bright nuclear foci were counted as Rad51 foci-positive cells. B and C, U2OS cells transfected with RFWD3 or control siRNA were treated with 1 mm HU for 16 h before the HU was removed. Cells were allowed to recover for indicated times and fixed for Rad51 immunostaining. Representative images are shown in B, and the statistical analysis is shown in C. The average number of Rad51 foci/cell was calculated among foci-positive cells. D, HeLa cells transfected with RFWD3 or control siRNA were treated with 10 Gy of IR and were allowed to recover for up to 24 h. The γH2AX as a marker for DSB was analyzed by immunostaining at 24 h. The arrows in siRFWD3-transfected cells point to cells that still express RFWD3, whereas arrowheads point to cells that have lost RFWD3 expression. E, cells were transfected and treated as in D but were analyzed by Western blotting. F, DNA damage sensitivity was measured by the colony formation assay in HeLa cells transfected with the indicated siRNAs. Two hundred cells were plated in triplicate and treated with 1 mm HU for 24 h or 2 Gy IR. Colonies were counted after recovering for 1 week.