FACS analysis of cell cycle parameters in DU145, DU145-Id4, PC3 and PC3-Id4 cell lines. The cells were serum starved for 24 hours in order to synchronize the cell cycle. The cells were then serum stimulated (10% BCS) in order to determine the effect of Id4 on cell cycle. A and D: Serum treated DU145 and PC3 cells respectively, B and E: Serum treated DU145-Id4 and PC3-Id4 cells respectively. The cell cycle analysis and the fraction of cells in each phase (G0-G1, S and G2/M, indicated as % cells) were determined using ModFit cell cycle analysis software. The data is representative of triplicate experiments. Of note is the number of cells (scale) represented on the Y-axis. C: Reverse Transcriptase polymerase chain reaction demonstrating the expression of Id4 in PC3 (lane 1) and PC3 cells stably transfected with Id4 expression plasmid (Lane 2). Weak Id4 expression was observed in PC3 cells whereas significantly higher expression was observed in PC3-Id4 cells (representative of 3 different PCR reactions). F: The ratio of cells in each phase. The ratio was calculated by dividing the % cells in Id4 expressing cells by parental cells in each phase of cell cycle. This calculation was used to normalize and compare the data between two cell lines. The error bars represent standard error of mean calculated from three different experiments (*: P > 0.05 and ***: P > 0.001).