FIG 4.

RhoB-deficient cells are defective for DSB repair by homologous recombination. (A) Substrate and strategy used to measure DSB-induced homologous recombination (26, 48). The pDR-GFP substrate contains two inactive genes coding for GFP under the control of a promoter (P). The 5′ gene is inactive because of the insertion of a cleavage site for I-SceI. The 3′ gene is inactive because it is deleted in both the 5′ and 3′ directions. When a DSB is produced by I-SceI, recombination between these two inactive genes (specifically gene conversion) restores a functional GFP coding sequence by either intrachromatid homologous recombination (represented) or unequal sister chromatid exchange (not represented). (B) RG37 cells stably expressing the pDR-GFP substrate were transfected with RhoB-targeting or nontargeting (control) siRNAs for 2 days and then transfected with an I-SceI plasmid for an additional 3 days. Cells transfected with Rad51-targeting siRNAs were used as a control for the pDR-GFP substrate. “No I-SceI” corresponds to cells transfected with an empty plasmid for 3 days. (Top) Percentages of GFP-positive recombinant cells determined by flow cytometry and normalized to the level of cells cotransfected with control siRNAs and I-SceI, which was set to a value of 1. Data shown are means ± standard errors of the means for five (with RhoB#1 siRNA) or three (with RhoB#2 and Rad51 siRNAs) independent experiments. ***, P < 0.001; **, P < 0.01 (by t test). (Bottom) Western blotting of RhoB and Rad51. α-Tubulin was the loading control. (C and D) HCT15/Chk2-WT cells were transfected with RhoB-targeting or nontargeting (control) siRNAs and left untreated or were treated with 1 μM CPT for 1 h. Rad51 foci were analyzed by immunofluorescence microscopy at 6 h post-CPT treatment (Release) (see protocol described in the legend to Fig. 3D). (C) Representative images. DAPI, 4′,6-diamidino-2-phenylindole. Bar, 10 μm. (D) Percentages of cells with at least five Rad51 foci. At least 200 cells were analyzed in each group (means ± standard errors of the means). (E) HCT15/Chk2-WT cells were transfected with RhoB-targeting or nontargeting (control) siRNAs, labeled with 30 μM BrdU for 30 min, and analyzed by flow cytometry. Numbers indicate percentages of BrdU-positive cells (means ± SD for three experiments). Unlabeled cells were used as negative controls for anti-BrdU staining. PI, propidium iodide. (F) Substrate and strategy used to measure DSB-induced end joining (49). The pCOH-CD4 substrate contains genes coding for the membrane antigens H2Kd, CD8, and CD4. The only expressed gene is H2Kd. CD8 is not expressed because it is in an inverted orientation, and CD4 is not expressed because it is too far from the promoter (P). Two cleavage sites for I-SceI are present in noncoding sequences, which are in direct orientation generating cohesive ends between the two sites. When two DSBs are produced by I-SceI, the internal fragment H2Kd/CD8 is excised, and rejoining of the DNA ends leads to the expression of the CD4 gene. (G) GC92 cells stably expressing the pCOH-CD4 substrate were transfected as described above for panel B, and percentages of CD4-positive cells were determined by flow cytometry using an anti-CD4 antibody. Nontargeting-siRNA-transfected cells treated with the DNA-PK inhibitor NU7026 (DNA-PKi) (10 μM) at the time of transfection with the I-SceI plasmid were used as a control for the pCOH-CD4 substrate. Data shown are means ± standard errors of the means for seven (with siRNA RhoB#1) or three (with siRNA RhoB#2 and the siRNA control plus the DNA-PK inhibitor) independent experiments. ns, nonsignificant (P = 0.15 [with siRNA RhoB#1] and P = 0.14 [with siRNA RhoB#2]); ***, P < 0.001 (by t test).