Figure 3.
Ectopic expression of cyclin E overcomes the PSM-RB G1 arrest. (A) Rat-1 cells were cotransfected with a CD20 expression plasmid (1 μg) and expression plasmids encoding the indicated proteins (PSM.7-LP or p16ink4a, 5 μg; cyclin E, 10 μg). Cells were harvested 48 hr post-transfection by trypsinization, stained with FITC anti-CD20, fixed in ethanol, and then stained with propidium iodide. The DNA content of at least 2500 CD20-positive, or the bulk CD20-negative, cells was determined by use of a FACScan cytometer with Cellfit software. Representative histograms and the cell cycle distributions from two independent experiments are shown. (B) Cells cotransfected with a puromycin-resistance plasmid (0.4 μg) and the indicated expression plasmids: 2.0 μg of PSM.7-LP plus 4.0 μg each of vector (lanes 1,4), cyclin E (lane 2), or SV40 T-Ag (lane 5), and vector alone (lane 3), were selected with puromycin for 72 hr and then harvested. From these selected cells, 15 μg of total protein was resolved by SDS-PAGE and cyclin A protein was detected by immunoblotting.