Figure 5.

Cyclin D1b overexpression in MCF-7 cells does not affect the DNA damage checkpoint. A, MCF-7 cells were infected with adenovirus encoding cyclin D1b, cyclin D1, and GFP (control) for 24 h. Cells were then treated with CDDP for 16 h. Total cell lysates were harvested and analyzed by immunoblotting with antibodies to RB, cyclin A, cyclin D1, cyclin D1b, and p21Cip1. Lamin B was used as a loading control. B, MCF-7 cells were treated as described in A of same figure. After treatment with CDDP, cells were pulse labeled with BrdUrd for 1 h, followed by BrdUrd staining and propidium iodide staining. Bivariate flow cytometry analysis of DNA synthesis (BrdUrd incorporation) and DNA content (PI) was performed. Shown are representative histograms of 20,000 gated events from two independent experiments. Quantitation of the percentage of BrdUrd-positive cells are from two independent bivariate flow cytometry experiments.