Figure 3
CDC27 inhibits p21-dependent arrest at the G1/S phase transition. (a) CDC27 overexpression in DLD1 stable cell lines and persistent suppression in HCT116 stable cell lines identified using real-time PCR and western blotting. Mean±S.D. of triplicate samples are shown. **P<0.01, ***P<0.001, Student’s t-test. (b and c) The indicated stable cell lines were subjected to cell cycle distribution analysis by flow cytometry. Images and qualification of the cell cycle distribution in three independent experiments are shown; *P<0.05, **P<0.01, Student’s t-test. (d and e) The HCT116 stable cell lines or DLD1 stable cell lines were synchronized by serum starvation for 24 h, and protein was harvested every 2 h after serum restoration. Western blotting analysis was used to detect changes in p21, p27 expression in the indicated cell lines. α-Tubulin was used as a reference control. (f) Cells were harvested at indicated time points after synchronization. Expression of p21 mRNA was analyzed by real-time PCR. Mean±S.D. of triplicate samples are shown. **P<0.01, ***P<0.001, Student’s t-test. (g) pGL3-p21 was transfected into both HCT116 and DLD1 stable cell lines, and cells were harvested at the indicated time after synchronization for luciferase reporter assays. Mean±S.D. of triplicate samples are shown. **P<0.01, ***P<0.001, Student’s t-test
