Fig. 4

Cyclin K is required for pre-RC assembly. a HCT116 cells were knocked down by indicated shRNA for 2 days and synchronized at M phase using procedure described in Fig. 2j, k. A portion of cells were used for protein blotting analysis (left panel), and the rest for FACS analyses (right panel). 0 h denoted cells collected immediately after release into fresh medium without nocodazole (800 ng/ml). b Sequential loading of prereplicative complex constituents (CDC6 followed by CDT1 and finally MCM2) onto chromatin in HCT116 cells from a were analyzed. Chromatin-bound PCNA marked S phase entry. Corresponding cell cycle phases were labeled at the bottom on basis of FACS analysis in c. c An aliquot of cells from b at each time point were analyzed by FACS analysis to demarcate each cell cycle phase. d Similar experiment as in b using HeLa cells. e Sequential loading of prereplicative complex constituents (CDT1 and MCM7) onto chromatin with or without CDK12 knockdown in HCT116 cells. f Rescue of pre-RC assembly defect by shRNA-resistant cDNA encoding cyclin K (K-R). cDNA was transduced into HCT116 cells, followed by transduction of shRNA 12 h later. vec., vector control. g Rescue of cell cycle defect by shRNA-resistant cyclin K in HCT116 cells. h An aliquot of HCT116 cells from f were used to determine the level of endogenous cyclin K transcript by qPCR analysis. Data are means ± SEM (n = 3) (***p < 0.001; Student’s t-test). All experiments were repeated at least three times and representative results are shown