Fig. 3.

SPIDR promotes homologous recombination. (A and B) SPIDR is required for RAD51 foci formation. U2OS cells were treated with IR (10 Gy) or CPT (1 μM) for 3 h before fixing and processed for RAD51 and γH2AX immunofluorescence. Representative RAD51 foci (IR treatment) are shown (A). (Magnification: 100×.) Quantification results were the average of three independent experiments and were presented as mean ± SEM (B). (C) Schematic representation of HR assay. (D) U2OS DR-GFP cells were transfected with the indicated siRNA and 24 h later were electroporated with an I-SceI expression plasmid. Forty-eight hours after electroporation, cells were harvested and assayed for GFP expression by FACS analysis. Results were the average of three independent experiments and were presented as mean± SEM (E). Knockdown efficiency was confirmed by immunoblotting. (F and G) Rescue of RAD51 foci formation by expression of a siRNA#1-resistant SPIDR cDNA. Immunostaining experiments were performed as described in Fig. 2F. Representative RAD51 foci (IR treatment) are shown (F). (Magnification: 100×.) Quantification results were the average of three independent experiments and are presented as mean ± SEM (G). (H) The exogenous SPIDR expression was confirmed by immunoblotting.