Figure S5.
DNA-PK activates AKT at the centrosomes. (A) AKT colocalized with centrosomal protein NEDD1. RPE-1 cells were coimmunostained with NEDD1 and AKT after different treatments. Colocalization of NEDD1 and AKT is shown in the Merge panel. Scale bar, 20 µm for labeled panel, 2 µm for magnified images. (B) pT308 AKT colocalized with centrosomal protein NEDD1. Depletion of AKT by siRNA abolished pT308 AKT on the centrosomes, indicating the specificity of anti-pT308 AKT antibody. Scale bar, 20 µm. (C) U2OS cells were treated with 2 Gy IR at 2 h after 200 nM leptomycin B pretreatment. The rate of microtubule polymerization was determined by microtubule regrowth assay at 2 h after IR treatment. Quantitation of microtubule length after IR treatment showed the rate of microtubule polymerization (n > 50). (D) NEDD1 depletion affected the NHEJ repair process and led to increased end resection of DSBs. The efficiency of NHEJ was indicated by γH2AX foci 8 h after IR treatment. The end resection was measured by RPA2 foci formation 2 h after IR treatment in HeLa cells. Quantitative results are shown (n > 50). (E) Representative images show the 53BP1 foci formation in shcon, shPCNT, or shNEDD1 cells after 5 Gy IR treatment in HeLa cells at indicated time points. Scale bar, 20 µm. (F) Upper: Representative images show chromosome breaks in shcon, shPCNT, or shNEDD1 HeLa cells without IR treatment. Chromosome DNA is stained with Hoechst, and there were no chromosome breaks in shPCNT or shNEDD1 cells without IR treatment. Lower: Quantitation of chromosome breaks per cell in shcon or shNEDD1cells with or without IR treatment (n > 30). ****, P < 0.0001; **, P < 0.01; ns, not significant. MT, microtubule; UT, untreated.

DNA-PK activates AKT at the centrosomes. (A) AKT colocalized with centrosomal protein NEDD1. RPE-1 cells were coimmunostained with NEDD1 and AKT after different treatments. Colocalization of NEDD1 and AKT is shown in the Merge panel. Scale bar, 20 µm for labeled panel, 2 µm for magnified images. (B) pT308 AKT colocalized with centrosomal protein NEDD1. Depletion of AKT by siRNA abolished pT308 AKT on the centrosomes, indicating the specificity of anti-pT308 AKT antibody. Scale bar, 20 µm. (C) U2OS cells were treated with 2 Gy IR at 2 h after 200 nM leptomycin B pretreatment. The rate of microtubule polymerization was determined by microtubule regrowth assay at 2 h after IR treatment. Quantitation of microtubule length after IR treatment showed the rate of microtubule polymerization (n > 50). (D) NEDD1 depletion affected the NHEJ repair process and led to increased end resection of DSBs. The efficiency of NHEJ was indicated by γH2AX foci 8 h after IR treatment. The end resection was measured by RPA2 foci formation 2 h after IR treatment in HeLa cells. Quantitative results are shown (n > 50). (E) Representative images show the 53BP1 foci formation in shcon, shPCNT, or shNEDD1 cells after 5 Gy IR treatment in HeLa cells at indicated time points. Scale bar, 20 µm. (F) Upper: Representative images show chromosome breaks in shcon, shPCNT, or shNEDD1 HeLa cells without IR treatment. Chromosome DNA is stained with Hoechst, and there were no chromosome breaks in shPCNT or shNEDD1 cells without IR treatment. Lower: Quantitation of chromosome breaks per cell in shcon or shNEDD1cells with or without IR treatment (n > 30). ****, P < 0.0001; **, P < 0.01; ns, not significant. MT, microtubule; UT, untreated.

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