FIG 2.

DDX1 facilitates HR-mediated DSB repair. (A) U2OS DR-GFP cells were transfected with scrambled siRNAs (control), DDX1 siRNA (si1 and si2), or RAD51 siRNA. After 72 h, cells were transfected with the same siRNAs along with an I-SceI expression construct by electroporation. GFP-positive cells were analyzed by flow cytometry. Relative HR efficiency was calculated by comparing percentages of GFP-positive cells in DDX1 siRNA- or RAD51 siRNA-treated cells versus control transfectants. (B) U2OS DR-GFP and U2OS DR-GFP HA-mDDX1 cells that stably express siRNA-resistant DDX1 were transfected with scrambled siRNAs (−) or DDX1 si1 (+). Relative HR efficiency was measured as described in for panel A. (C) Control and DDX1-depleted U2OS cells were treated with AG14361 at the indicated concentrations for 24 h. Cell survival was measured using the clonogenic assay. (D) Measurement of NHEJ efficiency in control and DDX1-depleted U2OS EJ5-GFP cells. Relative repair efficiency was calculated as described in for panel A. (E) Control and DDX1-depleted U2OS cells were exposed to 3 Gy IR. Cells were immunostained with anti-RAD51 antibody at 2 h post-IR. Bar, 10 μm. (F) U2OS cells and U2OS HA-mDDX1 cells that stably express siRNA-resistant DDX1 were transfected with scrambled (−) or DDX1 (+) siRNA. Numbers of RAD51 foci were analyzed 2 h after 3 Gy IR. For U2OS HA-mDDX1 cells, only cells that express HA-mDDX1 (positive for HA staining) were analyzed. (G) U2OS cells were transfected with scrambled or DDX1 siRNAs. Forty micrograms of lysates was resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Asterisk, residual actin signal. (H) U2OS cells were synchronized using a double thymidine block method. The numbers of RAD51 foci in control and DDX1-depleted cells were analyzed 2 h after 3 Gy treatment in S phase and G₂ phase. For all samples, n = 3, except for panel F, where n = 2; error bars are SEM. P values were calculated using Fisher's exact test for panels A and H and Student's t test for panel F.