Figure 6.

Cell cycle-dependent redistribution of cyclin D1 depends on the integrity of Thr-286. NIH-3T3 cells engineered to overexpress wild-type, Flag-tagged cyclin D1 (A) or Flag-tagged cyclin D1-(T286A) (B) were arrested in G₀ by serum deprivation and contact inhibition and then stimulated to synchronously enter the cell cycle. Cells were fixed at the indicated times after serum addition. The subcellular localization of D1 proteins was determined by staining with the cyclin D1 monoclonal antibody (top), and total cellular DNA was visualized with Hoechst dye (middle). In parallel, cells released from G₀ were stimulated to enter the cell cycle in the presence of BrdU to monitor S-phase entry. The percentage of cells incorporating BrdU at each time point is indicated below the panels. Because the levels of ectopic D1 expression were four- to eightfold above the endogenous background, a contribution of endogenous D1 to the staining pattern was negligible at the exposure chosen.