Fig 2: Inactivation of DNA-PKcs increases the ssDNA generation of DSB ends. a The production of ssDNA was visualized by immunostaining with anti-BrdU antibody. HeLa cells were pre-treated with 10 μM BrdU for 16 h, then cells were collected at 6 h after 10 Gy irradiation. The DNA-PKcs inhibitors Nu7441 and Nu7026 increased BrdU foci and ssDNA generation. b Quantitative determination of BrdU foci in 10 Gy-irradiated cells treated with or without Nu7441 or Nu7026. *p < 0.05. c The production of ssDNA was visualized by immunostaining with RPA32 antibody. The DNA-PKcs inhibitors Nu7441 and Nu7026 increased RPA32 foci and ssDNA generation. d Quantitative determination of RPA32/γH2AX double-labeled foci positive cells in 10 Gy-irradiated cells treated with or without Nu7441 or Nu7026. *p < 0.05. More than 50 cells were scored in each experiment

Fig 3: miR-205 enhances PCa xenograft response to radiation therapy. DU145 cells stably transfected with miR-205 or Neg vector (1 × 107) were implanted subcutaneously into the right flank of SCID mice. When tumors reached ~ 100 mm3, mice were randomly assigned to four groups (8 mice/group) and treated with 5 Gy single dose irradiation locally delivered to the tumor. (a) qRT-PCR showing miR-205 amount normalized to RNU-48 in Vec miR-205-tumours, compared to control ones. (b) Tumor growth volume (mm3) measured with a Vernier caliper on indicated days after cell injection. (c) Kaplan-Meier plot of mouse tumor growth to 1000 mm3

Fig 4: RBX1 promotes a decrease in EXO1 induced by ionizing radiation. a Effects of ionizing radiation on RBX1 and EXO1 protein expression. Western blotting analysis of RBX1, EXO1, Cullin1, and phosphorylated DNA-PKcs/s2056 in HeLa cells at the indicated time after exposure to 10 Gy. b The interaction between RBX1 and EXO1 was assessed in HeLa cells at 6 h after 10 Gy IR by immunoprecipitation assay. c, d After transfected with HA-Ub and SBP-Ub, the ubiquitination of the EXO1 protein was detected at 6 h after 10 Gy IR. To achieve the same content of EXO1 between cells treated with or without IR, proteasome inhibitor MG132 were added before ionization radiation. e Ionizing radiation increased the neddylation of Cullin1 protein at 2 h after 10 Gy IR. f HeLa cells were synchronized in G1 and G2 cells, respectively, and the neddylation of Cullin1 protein was detected at 2 h after exposed to 10 Gy IR. g The siRNA-NC and siRNA-RBX1 cells were exposed to 10 Gy IR and after 6 h, contents of EXO1 were evaluated with western blotting analysis

Fig 5: High-level ubiquitination mediated suppression of EXO1 in G1-phase cells. a HeLa cells were synchronized to different phases of the cell cycle by double-blockage of thymidine, and the cell phase distributions were monitored by flow cytometry analysis. b EXO1 protein was detected in the synchronized G1- and G2-phase HeLa cells by microscopic observation of immunofluorescent staining with anti-EXO1 antibody. Nuclei were stained with DAPI. c The expression levels of EXO1, RBX1, Cullin1, and phosphorylated DNA-PKcs-S2056 were assessed by western blotting analysis. Among them, phosphorylation of DNA-PKcs, is indicative of cells in different phase of cell cycle. d, e Ubiquitination of EXO1 protein was detected respectively in the synchronized G1- and G2-phase cells. After HeLa were transfected with HA-Ub and SBP-Ub plasmid for 48 h, the immunoprecipitation (IP) product obtained with the EXO1 antibody and S-bead agarose, were subjected to western blotting analysis with the anti-flag antibody and anti-EXO1 antibody separately. In order to achieve the same content of EXO1 for immunoprecipitation, when HA-Ub and SBP-Ub transfected cells were synchronized in G1- and G2-phase using a double thymidine block, cell were pre-treated with proteasome inhibitor MG132 for 2 h. f Hela were arrested at the G1/S boundary followed by a release into fresh media to allow cells to progress through the cell cycle. Cells were synchronized in G2 phase (7–9h) and G1 phase (12–20h) after TDR release. 10 μM MG132 was added in G1 and G2 cells for 2 h and EXO1 contents were detected. g Neddylation of Cullin1 protein was detected separately in the synchronized G1- and G2-phase cells. The immunoprecipitation (IP) product obtained with the Cullin1 antibody was subjected to western blotting analysis with anti-NEDD8 antibody. h The effect of neddylation on the stability of the EXO1 protein. Cells were pre-treated with the neddylation inhibitor MLN4924 or DMSO as a control for 8 h and then co-cultured with cycloheximide (CHX). EXO1 levels were assessed by western blotting analysis at the indicated times after CHX treatment