Fig 2: CRLDDB2 is a new E3 ubiquitin ligase of CDT2. a The protein level of CDT2 was accumulated when DDB2 was silenced. HCT116 cells were transfected with luciferase and DDB2 specific siRNAs for 48 h and subjected to Western blot. Actin was taken as loading control. Right panel: the relative protein levels of CDT2 and DDB2 were quantified by Gel-pro analyzer 4.0, and the P value was calculated by the two-side Student’s t-test (*** indicated P < 0.001). The error bars denoted standard deviation (SD). b Silencing of DDB2 accumulated exogenous CDT2. HCT116 cells were treated with indicated siRNAs for 18 h, and then transfected with pCMV10-3Flag-CDT2 by polyetherimide and cultured for another 40 h. The protein levels of CDT2 and DDB2 were analyzed by Western blot. Exogenous CDT2 was detected by anti-Flag antibody. The relative protein level of CDT2 was measured using Gel-pro analyzer 4.0 and plotted on the right panel. *** indicated P < 0.001. The error bar indicated SD. c DDB2 had negligible effects on CDT2 transcription. HCT116 cells were transfected with indicated siRNAs, and mRNAs were extracted at 24 h, 36 h and 48 h respectively. The mRNA levels of interested genes were measured by RT-qPCR. Two pairs of specific primers targeting to CDT2 were used to quantify mRNA level of CDT2. Student’s t-test was used to calculate P value (** indicated P < 0.01, *** indicated P < 0.001). The error bars denoted SD. d Down regulation of DDB2 prolonged the half-life of CDT2. HCT116 cells were transfected with specific siRNAs for 48 h, and then treated with 100 µg/mL CHX for indicated times. The protein levels of CDT2 and DDB2 were measured by specific antibodies. The relative protein level of CDT2 was plotted on the right panel. The error bars denoted SD. e DDB2 and CDT2 interacted mutually. HCT116 cells were harvested with lysis buffer after treated with Mg132 (10 µg/mL) for 4 h, and then immunoprecipitated with NRS (normal rabbit serum), anti-CDT2 and anti-DDB2 antibodies respectively. The protein levels of interest were detected by Western blot. f CRL4DDB2 complex promoted CDT2 polyubiquitination in vivo. HCT116 cells were co-transfected with pKH3-Ub and pCMV10-3Flag-DDB2 or pCMV10-3Flag (Vector) for 48 h, and lysed after MG132 treatment for 5 h. Cell lysate was immunoprecipitated with NRS or anti-CDT2 antibody, and immunocomplexes were analyzed with indicated antibodies. Immunoblots of whole cell extracts were shown at the bottom. g DDB2 promoted CDT2 degradation. HCT116 cells were transfected with pKH3 (Vector) or pKH3-DDB2 for 48 h, and the protein levels of interest were detected by Western blot. Bottom panel, the relative protein levels of CDT2 and DDB2 was quantified. The error bars represent SD

Fig 3: DDB2 mediated proteolysis of CDT2 may corelated with prognostic in cancer patients. a DDB2 promoted CDT2 proteolysis in Chang liver and CCC-HPF-1 cells. Chang liver and CCC-HPF-1 cells were treated with siRNAs of luciferase and DDB2 for 48 h, and harvested for Western blot. PCNA was taken as loading control. The relative protein levels of CDT2, CDT1 and DDB2 were normalized and plotted on the right panel. ** Denoted P < 0.01; *** denoted P < 0.001, and the error bars indicated SD. b CCC-HPF-1 cells were arrested in G1 phase after DDB2 silencing. FACS analysis of DNA contents in CCC-HPF-1 cells after treatment with siRNAs of luciferase and DDB2 for 48 h. c Expression of CDT2 and DDB2 in ovarian teratoma and breast cancer tissues detected by IHC. Scale bar: 50 µm and 100 µm. d The schematic model of CRL4DDB2 ubiquitin ligase regulates DNA replication initiation through degrading CDT2. In late M and G1 phase, the degradation of CDT2 mediated by CRL4DDB2 ubiquitin ligase stabilizes CDT1. Accumulation of CDT1 promotes recruitment of MCMs onto origins and assembly of pre-replication complex

Fig 4: Reducing the expression of DDB2 suppresses re-replication induced by CDT2 deficiency. a Silencing of DDB2 inhibited the formation of giant nuclei induced by CDT2 knockdown. HCT116 cells were transfected with indicated siRNAs for 48 h and fixed with 4% paraformaldehyde. DNA was stained by DAPI (4',6-diamidino-2-phenylindole). Scale bar: 50 µm. Right panel: the percentages of enlarged nuclei in a the error bars indicated SD. Significant difference was observed between CDT2 siRNA- and luciferase siRNA- or DDB2 + CDT2 double siRNA-treated cells, which was evaluated by the two-side Student's t-test (*** indicated P < 0.001). b FACS analysis of DNA contents in a. The percentages of DNA contents were presented in the right panel. The significant difference was observed between CDT2 siRNA- and luciferase siRNA- or DDB2 + CDT2 double siRNA-treated cells with 4N and > 4N DNA contents, which was evaluated by the two-side Student's t-test (*** indicated P < 0.001). c The efficacy of siRNAs in a and b were analyzed by Western blot. Right panel: The relative protein levels were measured using Gel-pro analyzer 4.0, and the P values were evaluated using the two-side Student's t-test (* denoted P < 0.05, ** denoted P < 0.01, *** denoted P < 0.001). The error bars indicated SD