Figure 5
Figure 5. The role of NHEJ DNA repair in nucleoplasmic bridge formation and BFB cycles in CML cells. (A) Plasmid-based assays for the SSA pathway of HR and NHEJ repair were performed in CML and normal CD34+ cells. (B) NHEJ repair in CML CD34+ cells (n = 3) and normal CD34+ cells (n = 5). Data are mean ± SEM of multiple experiments. *P < .05, ***P < .001 (comparing CML and normal CD34+ cells). (C) Expression of DNA-PKcs, Ku70, and Ku80 in normal CD34+ (n = 3) and CML CP CD34+ (n = 3) cells was measured by Western blotting. Representative results are shown. (D) Nucleoplasmic bridge formation in the BCR-ABL–positive cell line MBA-4 and its control cell line MYN. (E) Inhibition of Ku70 expression in MBA-4 cells by anti-Ku70 shRNA-expressing vectors was studied by Western blotting. A representative blot is shown. (F) Nucleoplasmic bridge formation in Ku70 knockdown and control MBA-4 CML cells at 24 hours (1 division) and 72 hours (3 or 4 divisions) after exposure to radiation. Data are mean ± SEM of 3 experiments. ***P < .0001 (comparing control-shRNA with Ku-shRNA1 and control-shRNA with Ku-shRNA2 at 24 and 144 hours after culture). (G) Nucleoplasmic bridges in MBA-4 cells after treatment with NU7026 were studied. Data are mean ± SEM of 3 experiments. A significant reduction in bridges was observed at 24, 72, and 144 hours compared with untreated controls (P < .0001). (H) siRNA-mediated inhibition of CtIP expression in MBA-4 cells was confirmed by Western blotting. A representative blot is shown. (I) Nucleoplasmic bridges were studied at 24, 48, and 72 hours after irradiation in MBA-4 cells after knockdown of CtIP with siRNA and compared with untreated cells and cells treated with control siRNA. A trend toward reduction of bridges was observed at all time points after CtIP knockdown, but the reduction was not significant at any of the time points. Data are mean ± SEM of 3 experiments. (J) Suppression of radiation-induced nucleoplasmic bridge formation in MBA-4 cells after treatment with increasing doses of dasatinib. At higher concentrations, dasatinib was inhibitory to growth of MBA-4 cells. At low doses of dasatinib, significant inhibition of nucleoplasmic bridge formation was seen.

The role of NHEJ DNA repair in nucleoplasmic bridge formation and BFB cycles in CML cells. (A) Plasmid-based assays for the SSA pathway of HR and NHEJ repair were performed in CML and normal CD34+ cells. (B) NHEJ repair in CML CD34+ cells (n = 3) and normal CD34+ cells (n = 5). Data are mean ± SEM of multiple experiments. *P < .05, ***P < .001 (comparing CML and normal CD34+ cells). (C) Expression of DNA-PKcs, Ku70, and Ku80 in normal CD34+ (n = 3) and CML CP CD34+ (n = 3) cells was measured by Western blotting. Representative results are shown. (D) Nucleoplasmic bridge formation in the BCR-ABL–positive cell line MBA-4 and its control cell line MYN. (E) Inhibition of Ku70 expression in MBA-4 cells by anti-Ku70 shRNA-expressing vectors was studied by Western blotting. A representative blot is shown. (F) Nucleoplasmic bridge formation in Ku70 knockdown and control MBA-4 CML cells at 24 hours (1 division) and 72 hours (3 or 4 divisions) after exposure to radiation. Data are mean ± SEM of 3 experiments. ***P < .0001 (comparing control-shRNA with Ku-shRNA1 and control-shRNA with Ku-shRNA2 at 24 and 144 hours after culture). (G) Nucleoplasmic bridges in MBA-4 cells after treatment with NU7026 were studied. Data are mean ± SEM of 3 experiments. A significant reduction in bridges was observed at 24, 72, and 144 hours compared with untreated controls (P < .0001). (H) siRNA-mediated inhibition of CtIP expression in MBA-4 cells was confirmed by Western blotting. A representative blot is shown. (I) Nucleoplasmic bridges were studied at 24, 48, and 72 hours after irradiation in MBA-4 cells after knockdown of CtIP with siRNA and compared with untreated cells and cells treated with control siRNA. A trend toward reduction of bridges was observed at all time points after CtIP knockdown, but the reduction was not significant at any of the time points. Data are mean ± SEM of 3 experiments. (J) Suppression of radiation-induced nucleoplasmic bridge formation in MBA-4 cells after treatment with increasing doses of dasatinib. At higher concentrations, dasatinib was inhibitory to growth of MBA-4 cells. At low doses of dasatinib, significant inhibition of nucleoplasmic bridge formation was seen.

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