E2F-1 target gene expression levels in asynchronously growing ψ-CRE cells overexpressing E2F-1/wt, E2F-1/411 and control (pX17). A. (Left Column) MRT-PCR of the total RNA of the ψ-CRE cells overexpressing E2F-1/wt , 411 and control was carried out as described in Materials and Methods and products were analyzed on a 1.5% agarose gel. The gels were stained with EtBr, and EtBr signals were analyzed by computer assisted densitometry. B. (Right Column) Results are presented as fold increases in expression of target gene for each cell line compared to control (pX17), which was given the value of 100. Values are mean ± S.E.M., n>3, * P< 0.05 compared to control (pX17) ψ-CRE cells. In both columns, from top to bottom, the genes analyzed were DHFR, B-myb, Cyclin D1, Cdc2, c-myc, TK and RB.

Cell cycle analysis of pX17, E2F-1/wt and E2F-1/411 overexpressing cells. A. Cells were starved in 0.5% FBS for 60 hours, at which time samples were taken and analyzed by flow cytometry. The histograms from pX17, E2F-1/wt and E2F-1/411 overexpressing cells are shown with the mean of cell numbers in G₀/G₁, S and G₂/M phases. B. Number of pX17, E2F-1/wt or E2F-1/411 overexpressing cells in G₀/G₁. G₀/G₁ values are mean ± S.E.M., n=4, * P< 0.05 compared to control (pX17) ψ-CRE cell line, u P< 0.05 compared to ψ-CRE E2F-1/wt cell lines. C. Cells were collected at various times after serum stimulation and the percentage of cells in S phase of the cell cycle was determined. Values are mean ± S.E.M., n=3.

Models for the E2F-1 and pRb roles for target gene expression. Models were designed from the data presented in this paper. While repressor E2F complex generic types is the most appropriate for the target genes including, DHFR, b-myb, cdc-2, Cyclin D1, C-myc and TK in cycling cells, the inhibited E2F complex is the most appropriate generic types of E2F complexes for the RB. Possible models were designed for individual genes in cells re-entering the cycle according to the results.

A. Overexpression of E2F-1/wt and E2F-1/411. Cell free extracts from an equivalent number of cells (5 X 10⁴) of E2F-1/wt and E2F-1/411 were analyzed by immunoblotting using anti-E2F-1 specific polyclonal antibody after separation on 10% SDS-PAGE. The position of the E2F-1 bands are indicated on the right. B. Co-immunoprecipitation of pRb and E2F-1. Cell free extracts from the ψ-CRE cell lines overexpressing E2F-1/wt, E2F-1/411, or control (pX17) were co-precipitated with a monoclonal antibody specific for E2F-1 and followed by western blotting with E2F-1 polyclonal antibody or pRB polyclonal antibody as described in Materials and Methods. C. Histograms of cell cycle analysis of pX17, E2F-1/wt and E2F-1/411 overexpressing asynchronously growing cells are shown with the mean of cell numbers in G₁, S and G₂/M phases. D. Number of pX17, E2F-1/wt and E2F-1/411 overexpressing asynchronously growing cells in G₁. G₁ values are mean ± S.E.M., n=4, * P< 0.05 compared to control (pX17) ψ-CRE cell line, u P< 0.05 compared to ψ-CRE E2F-1/wt cell lines. E. Cells were plated in soft agar medium and stained as described in Materials and Methods. Colonies greater than 50 μm in diameter were scored after three weeks of growth. Transformation frequency was expressed as the percentage of total plated cells that formed colonies. Values are mean ± S.E.M., n=5, * P< 0.05 compared to control (pX17) ψ-CRE cell line, u P< 0.05 compared to ψ-CRE E2F-1/wt cell lines.

E2F-1 target protein expression levels in asynchronously growing ψ-CRE cells overexpressing E2F-1/wt or E2F-1/411. Cell free extracts from an equivalent number of cells (5 X 10⁴) of E2F-1/wt and E2F-1/411 were analyzed by immunoblotting using specific antibody for the indicated individual protein after separation on 10% SDS-PAGE.

Roles of E2F-1 and pRb on target gene expression levels in synchronously growing ψ-CRE cells. A. (Left Column) MRT-PCR of the total RNA of control ψ-CRE cells (pX17) and ψ-CRE cells overexpressing E2F-1/wt and E2F-1/-411. The cells were starved and then stimulated back into the cell cycle at time 0 as described in Materials and Methods. Time points analyzed were time 0, 6 hours, 12 hours and 18 hours. Products were analyzed on 1.5% agarose gels. The gels were stained with EtBr, and EtBr signals were analyzed by computer assisted densitometry. B. (Right Column) Results from A are presented as fold increases in expression (y-axis) of each bar graph of target genes for each cell line compared to control (pX17), which was given the value of 100 at time 0. Values are mean ± S.E.M., n>3, * P< 0.05 compared to control (pX17) ψ-CRE cells at the same time point. In both columns, from top to bottom, the genes analyzed were DHFR, B-myb, Cdc2, Cyclin D1, c-myc, TK and RB.