PCNA is dispensable for degradation of CDT2 mediated by DDB2.
a Both wild-type DDB2 and PIP-mutant DDB2 promoted CDT2 degradation. Upper panel: the mutated sites in DDB2 PIP box. Middle panel: HCT116 cells were co-transfected with pKH3-CDT2 and pCMV10-3Flag-DDB2 or pCMV10-3Flag-DDB2-PIP-mutant plasmids for 40 h, and cell lysates were blotted by indicated antibodies. Lower panel: the relative protein levels of interest were measured by Gel-pro analyzer 4.0. The significance between experiment group (co-transfection of CDT2 and DDB2 or DDB mutant) and control group (only CDT2 transfection) was calculated by two-side Student’s t-test. *** Denoted P < 0.001. b DDB2 promoted degradation of both wild-type CDT2 or PIP mutant CDT2. Upper panel: the sequence of CDT2 PIP box and the amino acids deleted. Middle panel: HCT116 cells were co-transfected with pCMV10-3Flag-DDB2 and pCMV10-3Flag-CDT2 or pCMV10-3Flag-CDT2-PIP-mutant plasmids for 48 h, and cells were harvested for Western blot. GFP was co-transfected to indicate transfection efficiency. Proteins of interest were blotted by specific antibodies. Lower panel: the relative protein level of exogenous CDT2 was measured by Gel-pro analyzer 4.0 (lower panel). The P value was evaluated by the two-side Student’s t-test. *** Denoted P < 0.001. c Silencing of PCNA could not accumulate CDT2 protein. HCT116 cells were transfected with siRNAs specific targeting to PCNA, and harvested for Western blot analysis. The relative protein levels were normalized and plotted on the lower panel. *** Denoted P < 0.001. d Knockdown of PCNA could not up-regulate CDT2 at G1/S or S phase, but accumulated CDT1, p21 and SET8. HCT116 cells were transfected with luciferase and PCNA siRNAs, and synchronized at G1/S and S phase by double thymidine treatment. Proteins of interest were detected by indicated antibodies, and relative protein levels were measured by Gel-pro analyzer 4.0 (right panel). The P value was evaluated by the two-side Student’s t-test (** denoted P < 0.01; *** denoted P < 0.001). e Cell synchronization was confirmed by FACS. f PCNA has no effect on DDB2-mediated CDT2 degradation. HCT116 cells were transfected with luciferase, DDB2, PCNA and DDB2 + PCNA siRNAs for 60 h and total cell lysate was analyzed using indicated antibodies. The relative protein level of CDT2 was measured and plotted on the right panel. The P value was evaluated by the two-side Student’s t-test (** denoted P < 0.01; *** denoted P < 0.001). The error bars indicated SD