Figure 3. p27^CK– causes a cytokinesis defect.

(A) Multinucleation was quantified (500 cells counted/line, n = 3) in two lines of immortalized p27^–/– MEFs infected or not with p27^CK– and stained for β-catenin and lamin-A (original magnification, ×400). p27^CK– levels following retroviral infection were measured by immunoblot for p27, and β-tubulin levels were used as loading control. Lower graph: For each cell line, the proportion of mitotic cells in telophase and cytokinesis was determined in 50–100 mitotic figures; results are mean of 5 independent experiments. (B) Overexpression of p27^CK– in HeLa cells causes multinucleation. HeLa cells were transfected with either empty vector or a vector encoding p27^CK–. Transfected cells were visualized by p27 staining and co-labeled for β-catenin, and 600 p27-positive cells were counted (original magnification, ×400). Graphs show the average number of multinucleated cells in 3 independent experiments. Right graph: number of mitotic cells in telophase and cytokinesis observed in 100 mitotic figures (n = 4). In A and B, *P < 0.05, **P < 0.01, Student’s t test. (C) Number of mitotic cells in cytokinesis in immortalized p27^+/+, p27^–/–, and p27^CK– MEFs. One hundred mitotic figures were counted per genotype in 3 independent experiments. Results were analyzed by 1-way ANOVA with the Tukey-Kramer multiple comparison test; p27^+/+ versus p27^CK–, **P < 0.01. Error bars in A–C represent SEM. (D–H) Videomicroscopy of p27^+/+, p27^–/–, and p27^CK– MEFs undergoing mitosis. (D) Incidence of multinucleation caused by cytokinetic regression relative to the total number of mitotic figures recorded (p27^+/+: n = 181; p27^–/–: n = 117; p27^CK–: n = 243). (E) Nature of the defect causing multinucleation: multinucleation caused by reopening of the intercellular bridge as a percentage of the total number of multinucleation was determined for each genotype. (F–H) Examples of phase contrast videomicroscopy of mitotic p27^+/+ (F) and p27^CK– MEFs (G and H). Arrows indicate the intercellular bridge. In p27^CK–, cells undergo mitosis normally until the formation of intercellular bridge, which regresses to form binucleated cells. Original magnification, ×200.