Fig. 1.
Enforced expression of Cdc6 advances both reinitiation of S-phase progression and reactivation of Cdk2, whereas RNAi-mediated repression of endogenous Cdc6 expression retards recovery from S-phase arrest and reactivation of Cdk2. (A) Mid-S phase-synchronized original NRK and NRK-Cdc6#3 cells were treated with MMS and cultured with every 2-h cell harvests. The harvested cells were analyzed by flow cytometry (Upper) and assayed for Cdc6, S345-phosphorylated CHK1 (pCHK1), CHK1, p21, p53, p27KIP1, cyclin E (CycE), cyclin A (CycA), Cdk2, Cdk4, Cdk6, Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, Mcm7 by immuno-blotting and for the activity and amount of Cdk2 (Lower). The cell populations in each cell-cycle phase of the harvested cells were calculated from the cytometric patterns with the bundled software and are shown below the cytometric patterns. The factors not shown were unchanged during the time course and similar in levels between original NRK and the Cdc6 overexpresser. β-actin was used as loading control. (B) Logarithmically proliferating p27−/− MEF cells were transfected with the universal negative control RNA duplex or the Cdc6-specific duplex, 72 h later synchronized to mid-S phase, treated with MMS, and analyzed as in A. Cdk2 served as a loading control. (C) The nuclear extracts in A were analyzed for Cdc6 and p21 and assayed for the activity, amount, and phosphorylation states of Cdk2 and its associated proteins.