Fig. 3
Acute IR exposure elevated CDC6 protein levels by ubiquitin-proteasome pathways, and chronic IR elevated CDC6 protein levels by decreasing CDC6 phosphorylation-induced nuclear-cytosolic translocation. a CNE2 cells were exposed to 10 Gy X-ray radiation, and CDC6 protein was assessed 1, 24, 48, and 72 h after IR exposure. b The protein levels of CDC6 and Ki67 were assessed in CNE2 and CNE2-R cells. c The protein levels of CDC6 or Ki67 were analyzed by immunohistochemical staining in tumor specimens from NPC patients with partial or complete response (PR n = 31 or CR n = 23) to radiotherapy. d The protein expression was scored as intensity of staining multiplied by the percentage of positive cells, and the correlation of CDC6 and Ki67 were processed for statistical analysis. e The CDC6 levels were assessed in the low-grade glioma tissues with/o IR and chemotherapy. These data were derived from TCGA database, and the correlations of CDC6 and tumor progression were analyzed by using statistical analysis. f CNE2 cells were exposed to 10 Gy IR, and the protein levels of CDC6, Rb, pRb-s780, pRb-s795, pRb-s807/811, E2F1, and E2F3 were assessed 1, 24, 48, and 72 h after IR exposure. g IR exposure promoted CDC6 protein stability. CNE2 and CNE2-R cells were treated with or without IR for 40 h, followed by CHX treatment. CDC6 protein was tested 0, 2, 4, 6, 8 h after CHX treatment by western blot. The half-life of CDC6 protein was calculated according to the gray-scale analysis of bands. h CNE2 cells were exposed to 10 Gy IR and the protein levels of APCCDC20 and APCCDH1, SCFcyclin F and EMI1 were assessed 1, 24, 48, and 72 h after IR exposure. i The protein levels of APCCDC20 and APCCDH1, SCFcyclin F and EMI1 in CNE2 and CNE2-R cells were assessed. j The ubiquitination status of CDC6 protein between CNE2 and CNE2-R cells was compared. pcDNA3.1-HA-Ub was transfected into CNE2 and CNE2-R cells individually. 42 h after DNA transfection, the cells were treated with 25 μM MG132 for 6 h, and then cell lysates were subjected to immunoprecipitation with anti-CDC6 antibody and immunoblotted with anti-HA antibody. k The expression and subcellular localization of CDC6 protein were monitored by confocal microscopy. CNE2 and CNE2-R cells were fixed and incubated with CDC6 antibody and a second antibody labeled with Alexa Fluor 488 dye. The nuclei were labeled with DAPI. l The nuclear and cytoplasmic parts of CNE2 or CNE2-R cells were separated, and CDC6 protein in the nuclei or cytoplasm was tested. The expression value of CDC6 protein was scored by gray-scale analysis. The expression of CDC6 in the nuclei of CNE2 or CNE2-R cells was defined by total protein minus cytoplasmic protein. m The phosphorylation levels of CDC6, CDK2, Cyclin A, and Cyclin E assessed in CNE2 and CNE2-R cells by western blot. *P < 0.05, ** P < 0.01, *** P < 0.001
