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. 2011 Feb 11;30(6):1079–1092. doi: 10.1038/emboj.2011.27

Figure 3.

Figure 3

DNA-PK competes with DSB end resection. (A) Ku80 siRNA significantly increases RPA foci numbers post-IR. A549 cells were exposed to 1 Gy X-rays and RPA foci were enumerated as indicated either with or without BRCA2 siRNA. G2 cells were identified with CENP-F. Knockdown efficiency and typical images are shown in Supplementary Figure S5A. (B) Ku80 siRNA, DNA-PKcs siRNA or combined siRNA causes increased DSB end resection in A549 G2 cells. RPA and 53BP1 foci were enumerated after 1 Gy X-rays. Solid bars represent the number of RPA foci, and solid+hashed bars represent the total number of 53BP1 foci. The knockdown efficiencies are shown in the right panel. (C) Loss of Ku80 and DNA-PKcs increased IR-induced RPA retention in G2. RPA retention in G2 cells was analysed using α-RPA antibody after detergent extraction. Chromatin-associated RPA was detected as an Alex488 signal following FACS. APH alone does not induce detectable RPA signal by FACS (data not shown), although some level of RPA signal was detected by IF (Supplementary Figure S2). Percent of RPA positive was shown in the right panel. Error bars represent two independent experiments. (D) Increased IR-induced SCEs in Ku80 and DNA-PKcs double-knockdown G2 cells. IR-induced SCEs in G2 phase were analysed following 2 Gy X-rays. (E) A significant reduction of IR-induced BrdU foci formation in CHO cells expressing DNA-PKcs ABCDE S>A. In all, 20 μM BrdU was added 24 h before IR. Cells were extracted with 0.2% Triton for 1 min at 2 h after 2 Gy and stained with α-BrdU antibody without denaturation. (F, G) CHO cells expressing DNA-PKcs ABCDE S>A fail to form Rad51 foci at 1 h after 2 Gy X-rays in contrast to control cells and cells expressing the phosphorylation mimic, DNA-PKcs ABCDE S>D. G2 cells were identified by DAPI intensity using ImageJ. Similar results were obtained examining RPA foci (data not shown). DNA-PK expression levels in the V3 strains are shown in Supplementary Figure S5B. Further characterisation of the strains has been described previously (Chan and Lees-Miller, 1996; Cui et al, 2005; Meek et al, 2007).