CDKN3 is essential for normal mitosis. (A) Inducible knockdown of CDKN3 and MAD2 leads to multinucleation (arrows, 200× magnification). (B) Quantification of multinucleation in CDKN3 and MAD2 knockout cells. P < 0.0001 for both CDKN3 and MAD2 siRNA (one-way ANOVA; n = 5). Error bars represent mean values ± SEM. (C) Knockdown of CDKN3 and MAD2 decreases mitotic index in unsynchronized cells as shown by flow cytometry. P < 0.001 in one-way ANOVA; n = 6. Error bars represent mean values ± SEM. (D) Representative time-lapse frames of cells dividing 72 h after transfection with negative control siRNA (A) and CDKN3 siRNA (B). Note the shortened time between NEB (black arrows) and anaphase (red arrows) upon CDKN3 knockdown. Time-lapse images were taken on an automated imaging system (Pathway 855; BD) in a controlled environment (5% CO₂, 37°C) every 192 s (3.2 min) using laser autofocus and a 20× NA 0.75 objective lens (Olympus); only every other frame from relevant sequences is shown for simplicity. (E) Frequency distributions of anaphase times. Movies of individual cells in unsynchronized populations were followed manually frame-by-frame to detect NEB. 76 control cells and 137 siCDKN3-transfected cells were measured in three independent experiments. P < 0.0001 in t test. (F) Gallery of mitotic cells transfected with CDKN3 siRNA. Note the multipolar spindles, unattached chromosomes, multinucleation, and cleavage furrows cutting through partially decondensed chromosomes. (G) Quantification of abnormal mitoses in unsynchronized CDKN3 and MAD2 knockout cells. P < 0.0001 (one-way ANOVA; n = 5). Error bars represent mean values ± SEM.