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FIG 1. HeLa–CRISPR–DNA-PKcs cells have reduced levels of ATM and are sensitive to ionizing radiation (IR). (A) HeLa-CRISPR-control and HeLa–CRISPR–DNA-PKcs cells were harvested with trypsin EDTA and lysed in NETN buffer containing protease and phosphatase inhibitors (40). Fifty micrograms of total protein was run on SDS-PAGE and immunoblotted for DNA-PKcs, ATM, ATR, mTOR, Ku70, and Ku80 (loading control) as shown on the right. (B) Quantification from three separate experiments. Statistical significance was determined by one-way ANOVA. *, P < 0.05. (C) HeLa-CRISPR-control and HeLa–CRISPR–DNA-PKcs cells were either not irradiated (0) or irradiated with 1, 2, or 4 Gy of IR, and radiation sensitivity was determined using a clonogenic survival assay. The results represent the average number of colonies from three separate experiments. Statistical significance was determined by one-way ANOVA (*, P < 0.05). The error bars indicate standard deviations.
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FIG 2. Phosphorylation of Aurora A and PLK1 and accumulation of cyclin B1 are delayed and/or reduced in nocodazole-treated HeLa–CRISPR–DNA-PKcs cells. (A) HeLa-CRISPR-control and HeLa–CRISPR–DNA-PKcs cells were grown under asynchronous conditions (lane A) or incubated with nocodazole (40 ng/ml) for 6, 16, or 24 h, as indicated, harvested, and analyzed by SDS-PAGE and immunoblotted for markers of mitosis, as indicated on the right. The blots were probed for Ku80 as a loading control. The data are representative of at least 3 separate experiments. (B to J) Quantitation for HeLa-CRISPR-control cells and HeLa–CRISPR–DNA-PKcs cells. Total proteins were normalized to Ku80 as a loading control. Phosphoproteins were normalized to their respective total proteins. Results are shown in arbitrary units on the y axis. Statistical significance was determined using Student's t test. P values of <0.05 (*) were taken as statistically significant. The error bars indicate standard deviations.
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FIG 3. Nocodazole-induced expression of anillin and securin is reduced in HeLa–CRISPR–DNA-PKcs cells. (A) HeLa-CRISPR-control and HeLa–CRISPR–DNA-PKcs cells were incubated in the absence (0) or presence of nocodazole (40 ng/ml) and harvested after 6 or 16 h, as indicated. Whole-cell extracts were generated, run on SDS-PAGE, and immunoblotted as for Fig. 2. (B and D) Quantitation of anillin and securin expression from three separate experiments as for Fig. 2. Statistical analysis was carried out as for Fig. 2. (C) Whole-cell extracts (lanes E) from nocodazole-treated (40 ng/ml; 16 h) HeLa-CRISPR-control (lanes 1 to 3) or HeLa–CRISPR–DNA-PKcs (lanes 4 to 6) cells were run on SDS-PAGE either directly (lanes E) or after incubation with 0.5 μl lambda phosphatase at 30°C for 10 min (lanes P) or incubated without phosphatase (mock treated; lanes M). Extracts were run on SDS-PAGE and immunoblotted for anillin and Ku80, as shown. The error bars indicate standard deviations.
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FIG 4. A549 cells with CRISPR depletion of DNA-PKcs have reduced and/or delayed nocodazole-induced upregulation of anillin, cyclin B1, and securin and reduced phosphorylation of Aurora A, PLK1, and TPX2. (A) A549-CRISPR-control, A549–CRISPR–DNA-PKcs, and A549-CRISPR-ATM cells were treated with nocodazole for 6 or 16 h, and then extracts were run on SDS-PAGE and analyzed by Western blotting as for Fig. 2. (B to M) Quantitation from 3 separate experiments. (B, D, E, G, H, J, K, L, and M) Proteins were normalized to Ku80. (C, F, and I) Phosphoproteins were normalized to their respective total proteins. Both are shown in arbitrary units (y axis). Statistical analysis was carried out by one-way ANOVA, with P values of <0.05 considered statistically significant. *, statistically significant difference between control and CRISPR-DNA-PKcs cells; #, statistical significance between CRISPR-DNA-PKcs and CRISPR-ATM cells. The error bars indicate standard deviations.
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FIG 5. Depletion of PP6 does not rescue nocodazole-induced downregulation of mitotic proteins in DNA-PKcs-deficient HeLa cells. (A) HeLa–CRISPR–DNA-PKcs cells were transfected with either scrambled control siRNA (siRNA-sc) or siRNA to PP6c (siRNA-PP6c) (100 nM), and after 56 h, the cells were treated with nocodazole (40 ng/ml) and harvested immediately (0) or after 3, 6, or 16 h, as indicated. Extracts were probed for mitotic proteins as described above. The Western blots are representative of 3 separate experiments. (B to K) Quantitation shown as in Fig. 2 and 4. The asterisk on the securin Western blot indicates a nonspecific band. siRNA-sc, scrambled siRNA control. The error bars indicate standard deviations.
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FIG 6. Depletion of PP6 does not rescue nocodazole-induced downregulation of mitotic proteins in DNA-PKcs-deficient A549 cells. The experiment was carried out exactly as for Fig. 5, but A549–CRISPR–DNA-PKcs cells were used. The asterisk on the securin blot indicates a nonspecific band. The error bars indicate standard deviations.
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FIG 7. Inhibition of APC/C with proTAME rescues expression and phosphorylation of mitotic proteins in nocodazole-treated DNA-PKcs-deficient HeLa cells. HeLa-CRISPR-control and HeLaâCRISPRâDNA-PKcs cells were preincubated with apcin (48âμM), proTAME (12âμM), or DMSO control for 1 h, and then nocodazole was added to 40âng/ml and cells were harvested after a further 16 h. Representative Western blots (A) and quantitation from 3 separate experiments (B to M) are shown as in Fig. 5 and 6. (A, B, C, D, E, G, H, J, K, and M) Proteins were normalized to Ku80. (F, I, and L) Phosphoproteins were normalized to their respective total protein controls. The error bars indicate standard deviations. *, Pâ<â0.05; ns, not significant.
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FIG 8. Inhibition of APC/C with proTAME rescues expression and phosphorylation of mitotic proteins in nocodazole-treated DNA-PKcs-deficient A549 cells. Experiments were carried out exactly as for Fig. 7, except that A549-CRISPR-control and CRISPRâDNA-PKcs cells were used. The error bars indicate standard deviations. *, Pâ<â0.05; ns, not significant.
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FIG 9. Model for the role of DNA-PK in regulation of APC/C activity in nocodazole-treated cells. (A) Nocodazole causes microtubule depolymerization, preventing attachment of kinetochores to the mitotic spindle. In controls cells, this leads to activation of the SAC; Cdc20 is sequestered by the MCC; APC/C is not activated; and levels of anillin, securin, cyclin B1, and other proteins (not shown) rise. (B) In DNA-PKcs-deficient cells, Cdc20 and Cdh1 interact with and activate the APC/C, causing degradation of mitotic proteins, such as anillin, securin, and cyclin B1, even in the presence of unattached kinetochores. (C) In DNA-PKcs-deficient cells treated with ProTAME, APC/C-mediated degradation of anillin, securin, cyclin B, and other proteins (not shown) is inhibited, causing levels of proteins to rise.
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