(A): Doubling time (in days, White bars) and rate of proliferation (Black bars) of DU145, DU145-CMV and DU145-Id4 cells line in culture. All the cell lines have matched passages. The DU145 cells were either transfected with no plasmid (DU145), CMV vector alone (DU145-CMV/EV) or pCMV-Id4 (DU145-Id4). White Bars: The average doubling time of DU145-Id4 cells as compared to DU145 and DU145-CMV. The data is an average cell count from four passages. The doubling time was calculated by counting days required for the number of plated cells (usually at 40% confluence) to reach confluence (80%). For DU145-Id4 cells, this average is from passage 13–16. A highly significant increase in the doubling time (approximately 2.5 fold) (P < 0.001) suggests that Id4 over-expression leads to a decrease in proliferation. Black Bars: 3H thymidine incorporation assay. 3H thymidine incorporation (CPM) was evaluated as a measure of rate of proliferation. The counts per minute (CPM) of incorporated 3H-thymidine was normalized to total DNA. The data represents CPM/ug DNA of cell lines at passages 14 and 15. The data is represented as mean ± SEM of three experiments performed in triplicates. *: P < 0.001. (B) FACS analysis of cell cycle parameters in DU145 and DU145-Id4 cell lines. The cell cycle profile showing fraction of cells in each phase of the cell cycle in DU145-Id4 and DU145 cells is shown. The data is representative of at least three experiments. The inset shows the fraction of cells in sub-G0 phase (normalized to 10,000 cells). The cells in sub-G0 phase have low DNA content possibly due to apoptosis. The mean ± SEM of three experiments performed in triplicate is shown (*: P < 0.001).