Phenotypic analysis of conditional INCENP knockout cell line. (A) Immunofluorescence images showing INCENP^ON and INCENP^OFF cells stained with specific antibodies recognizing endogenous INCENP (green; panels 3 and 7), α-tubulin (red; panels 2 and 6), and DAPI for DNA (blue; panels 4 and 8). After incubation with doxycycline for 26 h, endogenous INCENP was hardly detected (INCENP^OFF; panel 7). Panels 9–16 show immunofluorescence images of H3S10ph (green; panels 10 and 14) and H3S28ph (red; panels 11 and 15) plus DAPI for DNA (blue; panels 12 and16) in INCENP^ON (panels 9–12) and INCENP^OFF (panels 13–16) cells. Merged images are shown in panels 1, 5, 9, and 13. INCENP^ON and INCENP^OFF cells were harvested and fixed at the same time. Images were acquired using the same microscope settings for all experiments. Bar, 5 µm. (B) Mitotic indices of wild-type, INCENP^ON, and INCENP^OFF cells. (C) Scoring of mitotic cells in late prometaphase and metaphase where cells are bioriented and two spindle poles are on opposite sides. (D–G) Percentage of mitotic cells in prometaphase or metaphase (D), anaphase (E), telophase (F), or cytokinesis (G) is shown. (H–J) Percentage of binucleated cells (H), multinucleated cells (I), and mitotic cells with multipolar spindles (J) is shown. Error bars indicate SD.