A: Percentage of centrosome amplification in wild-type () or E2F1^−/− (■) MEFs from two independent litters (L.1 and L.2).
B: Percentage of centrosome amplification in wild-type () or E2F2^−/− (■) MEFs from two independent litters (L.1 and L.2).
C: Percentage of centrosome amplification in wild-type () or E2F4^−/− (■) MEFs from two independent litters (L.1 and L.2).
D: Percentage of centrosome amplification in wild-type () or E2F5^−/− (■) MEFs from two independent litters (L.1 and L.2). Centrosomes from early passage (p 2) MEFs were stained with a primary γ-tubulin antibody and a rhodamine-conjugated secondary antibody.

A: Western blot analysis of E2F3^−/− (−/−) and E2F3^f/f (f/f) MEFs infected with control (−) or Cre-expressing (+) retroviruses. Arrows indicate endogenous E2F3a and E2F3b proteins.
B: Fold induction in centrosome amplification in two independent litters (L.1 and L.2) of wild-type () or E2F3^f/f (■) MEFs. Cells were infected with control (−) or Cre-expressing (+) retroviruses as indicated, and centrosomes were stained with a primary antibody against γ-tubulin and a rhodamine-conjugated secondary antibody.

A: Western blot analysis of protein lysates from two litters (L.1, L.2) of wild-type or E2F3^−/− cells infected with control (C), myc-E2F3a-expressing (3a), myc-E2F3b-expressing (3b), or with both myc-E2F3a- and myc-E2F3b-expressing (3a/b) retroviruses. Cell lysates were subjected to SDS-PAGE, transferred unto PDVF membranes, and probed with an antibody that recognizes the myc epitope of the myc-tagged E2F3a and E2F3b proteins.
B: Fold induction of centrosome amplification in wild-type () or E2F3^−/− (■) ells. MEFs were infected with retroviruses as in (A), and centrosomes were stained with a primary antibody against γ-tubulin and a rhodamine-conjugated secondary antibody.
C: Fold induction of centrosome amplification in wild-type () or E2F3^−/− (■) cells infected as in (A), except that parallel plates were infected with retroviruses expressing HA-hE2F1 (1) or HA-hE2F2 (2).

A: Coimmunostaining of γ-tubulin (middle panels) and nucleophosmin B (right panels) in cells cultured under serum-starvation conditions.
B: Percentage of nucleophosmin B-positive unduplicated centrosomes in wild-type () or E2F3^−/− (■) cells under serum starvation conditions.
C: Percentage of cells with nucleophosmin B-positive centrosomes in the total population of wild-type () or E2F3^−/− (■) cells cultured under serum starvation.
D: Wild-type and E2F3^−/− cells expressing GFP-centrin-2. Shown from top to bottom: a wild-type cell with an unduplicated centrosome, prior to the separation of centrioles; an E2F3^−/− cell with a separated centrosome; an E2F3^−/− cell with multiple, separated centrioles. The left panels show the corresponding DAPI-stained nuclei. White arrows indicate individual centrioles while the red arrow indicates a separated centriole associated with a nascent centriole.
E: Top panel, percentage of wild-type () or E2F3^−/− cells (■) containing one (solid bars) or ≥2 separated centrosomes. Bottom panel, percentage of wild-type () or E2F3^−/− cells (■) containing unduplicated (solid bars), ≥2 separated and unduplicated (stripes), or ≥ 2 separated and duplicated centrosomes (dotted) in density-arrested conditions. Centrioles were visualized using GFP-centrin-2, and the calculated percentages are based on the results from counting 100 cells. Shown in parenthesis are the percentage of BrdU-positive cells in the population.

A: Spindle abnormalities were detected by coimmunostaining the mitotic asters with a primary antibody against α-tubulin and an ALEXA-488-conjugated secondary antibody (shown in green), and the centrosomes with a primary antibody against γ-tubulin and a rhodamine-conjugated secondary antibody (shown in red). The left panel shows a wild-type cell in metaphase with two centrosomes and a normal, bipolar mitotic spindle; the middle and right panel show two E2F3^−/− cells with multipolar mitotic spindles.
B: Percentage of wild-type () or E2F3^−/− (■) mitotic cells with abnormal multipolar spindles.
C: Percentage of wild-type () and E2F3^−/− (■) cells containing micronuclei. MEFs derived from three wild-type and four E2F3^−/− embryos from one litter are presented. The results were derived from counting 2000 cells in interphase per group (from duplicate plates).
D: Analysis of ploidy in early passage (p 2) wild-type (left panels) and E2F3^−/− (right panels) MEFs.
E: Analysis of ploidy in late passage (p 7) wild-type (left panel) or E2F3^−/− (right panel) MEFs. At least 50 chromosome spreads were counted per group. Each pair of panels represents wild-type and E2F3^−/− MEFs from the same litter. The four pairs presented are from independent litters.

A: Flow cytometric analysis of various early passage mouse embryonic fibroblasts (MEFs). Passage 2 wt or E2F3^−/− MEFs were processed as described in Experimental Procedures and analyzed by flow cytometry. Shown are the flow cytometric analyses of cells, showing their respective DNA contents (2N, 4N, or 8N).
B: Quantitative analysis of the percentage of passage 2 wt or E2F3^−/− MEF cells with the indicated DNA contents. Values indicate cells with 2N (■), 4N (), or 8N (□) DNA contents ± the standard deviation between four embryos from independent litters.
C: Flow cytometric analysis of control, nocodazole, or DCB and nocodazole-treated MEFs. Passage 5 wt, p53^−/−, rb^−/−, or E2F3^−/− MEFs were left untreated or were treated with nocodazole for 72 hr, or with DCB for 36 hr followed by nocodazole treatment for 36 hr. Shown are cells with a 2N, 4N, 8N, or 16N DNA content.

A: Immunohistochemical analysis of centrosomes from wild-type and E2F3^−/− cells using a primary γ-tubulin antibody and a rhodamine-conjugated secondary antibody (right panel); the left panel shows the same cells stained with DAPI.
B: Percentage of centrosome amplification in early passage (p 2) MEFs of three independent litters (L.1–L.3) of wild-type () and E2F3^−/− cells (■). The first and second litters include three wild-type and four E2F3^−/− embryos each; the third litter includes one wild-type and one E2F3^−/− embryo. Centrosomes were stained with a monoclonal antibody against γ-tubulin and a rhodamine-conjugated secondary antibody; the results represent the percentage of cells with three or more centrosomes.
C: Immunohistochemical analysis of the centrosome status of wild-type and E2F3^−/− cells using a primary antibody against pericentrin and a rhodamine-conjugated secondary antibody (right panel); the left panel shows the DAPI-stained mitoses.
D: Percentage of cells with three or more centrosomes from three wild-type and three E2F3^−/− embryos from two independent litters. Shown are the percentages resulting from staining wild-type cells () or E2F3^−/− embryos (■) with γ-tubulin (solid bar) or pericentrin (striped bar). For all the graphs presented in this section, we counted >200 cells per embryo from two different experiments.

A: Western blot analysis showing Cyclin E (top panel) or Cdk2 (as a loading control, bottom panel) protein levels in proliferating and starved wild-type and E2F3^−/− cells.
B: Western blot analysis showing Cyclin E protein levels in serum-starved wild-type, E2F1^−/−, E2F2^−/−, E2F3^−/−, E2F4^−/−, and E2F5^−/− cells. The bottom panel shows α-tubulin levels, used as loading controls. The lysate from E2F3^−/− MEFs presented were derived from a different litter than the ones shown in (B).
C: Relative activity of cyclin E-dependent kinase in protein lysates derived from starved and proliferating wild-type and E2F3^−/− cells, using histone-1 (H1) as a substrate.
D: Western blot analysis of isolated GST-tagged wt nucleophosmin B (GST-NPM/wt) or a GST-tagged mutant nucleophosmin, in which the threonine 199 site was changed to alanine (GST-NPM/T199A). The left panel shows the coomassie-stained gel to show the equal loading of protein, and the right panel a Western blot probed with an antibody against phospho-T199 nucleophosmin B.
E: Western blot analysis of nuclear and cytoplasmic protein fractions from serum-starved wt or E2F3^−/− MEFs. The Western blot was probed with the antibody described in (D). To ensure that equal loading was achieved, the same membrane was probed with α-tubulin.
F: wt and E2F3^−/− cells were cultured under serum starvation conditions and released from the G1 block by serum addition. Nuclear or cytoplasmic fractions were obtained 0, 4, and 16 hr after serum addition. The Western blot was probed with the same antibody used in (D) and (E). As a loading control, we probed the same membrane with an antibody against total nucleophosmin (NPM).