Idas RNAi prevents normal cell cycle progression. A, quantification of Idas mRNA and protein levels in HeLa cells after Idas RNAi, relative to control RNAi, by real time PCR (graph) and Western blot analysis using an Idas-specific antibody (inset, hIdasAb1) 48 h post-transfection. B and C, HeLa cells were transfected with Idas siRNA or control siRNA and 48 h post-transfection pulsed with BrdU for 30 min, fixed, and immunolabeled with anti-BrdU and anti-Geminin or anti-Cdt1 antibodies. DNA was stained with Hoechst. Quantification of cells positively stained for BrdU, Geminin, and Cdt1 in Idas siRNA-treated normalized to control-treated HeLa cells is shown in B. For each experiment, the % positive cells in control-treated cells was set to 1. -Fold increase/decrease is shown as a mean of three independent experiments, with S.D. More than 400 cells were measured for each experiment. Representative images are shown in C, with % positive cells ±S.D. indicated in each image. D, HeLa cells were treated with Idas or control siRNA and 48 h post-transfection were treated with BrdU for a total of 24 h. Time points were collected at 1, 2, 4, 6, 8, 10, 12, 16, 18, and 24 h after BrdU addition. Cells were fixed and stained with anti-BrdU antibody. DNA was stained with Hoechst. The BrdU labeling index was plotted against the BrdU pulse time. E, HeLa cells were treated with Idas or control siRNA, and 48 h post-transfection cells were treated with BrdU for 30 min, then BrdU was washed out, and cells were left in a BrdU-free medium. After BrdU removal, time points were collected at 30 min and 4, 8, and 12 h. Cells were fixed and immunolabeled with anti-BrdU and anti-Cdt1 antibodies. DNA was stained with Hoechst. The percentage of all cells scoring positive for BrdU (upper) and the percentage of BrdU-positive cells that show mitotic nuclear morphology (middle) or are positive for Cdt1 (bottom) are plotted at each time point to determine progression into Mitosis and G1 phase, respectively.