Figure 4: TRAIP is required for DNA-damage checkpoint and homologous recombination repair.
From: TRAIP/RNF206 is required for recruitment of RAP80 to sites of DNA damage
(a) Counts of γH2AX foci at various time points after 1-Gy ionizing radiation in HeLa cells transfected with indicated siRNAs or combination of siRNA and siRNA-resistant WT TRAIP, TRAIP-D2 or -D6 expression vector. (b) Comparison of homologous recombination repair capacity in DR-GFP U2OS cells transfected with indicated siRNAs or combination of siRNA and siRNA-resistant WT TRAIP, TRAIP-D2, -D6 or -A6. (c,e) 293T cells were transfected with indicated siRNA or combination of siRNA and siRNA-resistant WT TRAIP, TRAIP-D2, -D6 or -A6. After 48 h, transfected 293 T cells were exposed to 10-Gy of ionizing radiation, followed by staining with anti-BRCA1 (c), 53BP1 (e) or γ-H2AX antibodies. DAPI was used as an indicator for the nucleus. The percentage of cells with positive BRCA1 or 53BP1 foci from 100 counts was determined. (d) Non-homologous end joining repair assay was carried out in triplicates using U2OS cells harbouring NHEJ reporter (EJ5-GFP). The reporter cells were transfected with indicated siRNA and the percentage of GFP-positive cells were analysed using flow cytometry. (f) G2/M checkpoint in HeLa cells transfected with siRNAs or combination of siRNA and siRNA-resistant WT TRAIP, TRAIP-D2, -D6 or -A6. The transfected cells were exposed to 0 or 2 Gy of ionizing radiation and subjected to staining with antibody to phosphorylated histone H3 and propidium iodide. The percentages of mitotic cells were determined using flow cytometry. (g) Radiation sensitivity in HeLa cells transfected with siRNAs or combination of siRNA and siRNA-resistant WT TRAIP, TRAIP-D2, -D6 or -A6. These experiments were performed in triplicate, and the results represent the average of three independent experiments in each comparison. Error bars indicate the s.d.
