Fig. 6. Inhibition of new Ciz1 synthesis restrains entry to S phase. (A) Left: siRNAs that target Ciz1 transcripts at two sites (see Fig. 2A) were individually applied to cycling 3T3 cells and cell number was monitored at the indicated times. Right: images of cell populations at 16 and 40 hours after transfection with siRNA 8 (red outline) or mock treated cells (blue outline). (B) The proportion of cells that incorporate BrdU into DNA (green) is significantly decreased in cells treated with Ciz1 siRNAs 4 and 8, compared to GAPDH siRNA, 48 hours after treatment. Histogram shows average results from four independent experiments. (C) The number of nuclei with detergent-resistant Mcm3 and PCNA (green) increases in populations treated with Ciz1 siRNA 4/8, compared with mock-treated controls. All nuclei were counterstained and are shown in pseudocolour (red). (D) Quiescent 3T3 cells were stimulated to re-enter the cell cycle (Coverley et al., 2002) with and without exposure to Ciz1 siRNA 4/8 or GAPDH siRNA, and pulse labelled with BrdU at the indicated times to reveal the proportion of cells in S phase. (E) Quiescent 3T3 cells were stimulated to re-enter the cell cycle and harvested at the G1-S transition (18 hours, black) or after S phase (36 hours in the presence of nocodazole, red) with and without exposure to Ciz1 siRNA 4/8. (F) Detergent-soluble and -insoluble fractions of endogenous Ciz1 protein in approximately 1x10⁵ cells after mock treatment or treatment with Ciz1 siRNAs 4/8, detected with anti-Ciz1 1793. (G) To focus on newly synthesized Ciz1, expression of ectopic GFP-ECiz1 was monitored in whole cell extracts made from approximately 8x10³ cells, in the presence and absence of Ciz1 siRNA 4/8 using anti-Ciz1 1793. Expression levels in extracts prepared 22 hours after transfection are shown. For F and G band intensities were quantified (shown in white), normalised against actin levels and expressed as a percentage.