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. 2020 Mar 31;23(4):101027. doi: 10.1016/j.isci.2020.101027

Figure 5.

Figure 5

MRE11 Nuclease and UBC13-Mediated Ubiquitin Signaling Are Required for Efficient Repair of IR-Induced Dirty DSBs in the G1 Phase

(A) Repair rate of DSBs induced by AsiSI restriction enzyme in G1-phase TK6 cells. Cells expressing AsiSI fused with estrogen receptor (ER) were treated with 4-OHT for 4 h for DSB induction. We analyzed γH2AX foci in cyclin A-negative cells after 4-OHT was removed (time 0 h). We subtracted the average number of foci in the 4-OHT-untreated cells from the average number of foci in the 4-OHT-treated cells. Values correspond to the percentage of unrepaired DSBs relative to the value at time 0 h, set to 100%. Error bars were plotted for standard deviation (SD) from three independent experiments. More than 50 G1 cells (cyclin A negative) were analyzed for each experiment. Single, double, and triple asterisks indicate p = 1.9 × 10−5, p = 2.9 × 10−5, and p = 4.3 × 10−5, respectively, calculated by Student's t test. Box plots of γH2AX foci at the indicated time points (0, 1, 2, and 4 h) are shown in Figure S5A.

(B–E) Repair rate of DSBs induced by IR (1 Gy) in G1-phase TK6 and HCT116 cells. Average number of γH2AX foci in cyclin A-negative cells was counted at the indicated time points. We subtracted the average number of foci in IR-untreated cells from the average number of foci in IR-treated cells. Values correspond to the percentage of unrepaired DSBs relative to the value at time 0.3 h, set to 100%. Error bars were plotted for standard deviation (SD) from three independent experiments. In (B), p values were 2.0 × 10−4 (shUBC13 versus wild-type), 2.1 × 10−3 (shBRCA1 versus wild-type), 1.9 × 10−5 (MRE11-/H129N versus wild-type), 9.5 × 10−5 (LIG4−/−/MRE11-/H129N versus wild-type), 4.1 × 10−4 (EXD2−/− versus wild-type), 3.6 × 10−2 (ARTEMIS−/− versus wild-type), 6.3 × 10−6 (EXO1−/− versus wild-type), and 1.7× 10−4 (shUBC13/MRE11-/H129N versus wild-type), calculated by Student's t test. Box plots of γH2AX foci at the indicated time points (0, 0.3, and 6 h) are shown in Figure S5D. In (C), asterisks indicate p = 1.3 × 10−4 (single) and p = 7.9 × 10−5 (double), calculated by Student's t test. Box plots of γH2AX foci at the indicated time points (0, 0.3, and 6 h) are shown in Figure S6D. In (D), asterisks indicate p = 5.1× 10−4 (single) and p = 3.8 × 10−2 (double), calculated by Student's t test. Box plots of γH2AX foci at the indicated time points (0, 0.3, and 6 h) are shown in Figure S5F. In (E), asterisks indicate p = 1.2× 10−2 (single) and p = 3.2 × 10−3 (double), calculated by Student's t test. Box plots of γH2AX foci at the indicated time points (0, 0.3, and 6 h) are shown in Figure S5H.