FIG. 6.

E2F target genes are expressed in QKO cells. (A) cDNA prepared for the experiment shown in Fig. 5 was also used to determine the expression levels of E2F target genes in proliferating TKO versus QKO cells. In contrast to the results shown below, this graph demonstrates repression (n-fold) of gene expression in the cre-treated cells relative to that in the cells infected with control virus. (B) Induction of S phase and real-time PCR analysis of E2F target genes in TKO and QKO cells. Cells were synchronized by serum starvation, stimulated by the addition of serum, and harvested at the indicated time points. Total RNA was extracted from the cells and subsequently used to produce cDNA and determine the levels of the indicated E2F target genes. In order to determine the timing of S-phase entry in these cells relative to that of gene expression, the incorporation of 5-bromodeoxyuridine was also analyzed. BrdU was added to the plates at the same time as the serum, and harvesting took place at the zero time point as well as at the 8-hour time point. A total of 500 DAPI-stained nuclei from each cell line was counted, and the percent positive for BrdU incorporation is shown. Percent BrdU incorporation is indicated by the dashed lines, and solid lines represent induction (n-fold) of target genes. Symbols: □, cells treated with control virus; •, cre-treated cells.