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. 2016 Jul 20;2(7):e00131. doi: 10.1016/j.heliyon.2016.e00131

Fig. 13.

Fig. 13

Overexpression of INCENP-S894A mutant promotes multipolar cell division after release from nocodazole arrest. (A) Experimental strategy for characterization of INCENP mutants in late mitosis. Phosphorylation of S894-INCENP is induced by treatment of cells with nocodazole (Noc). INCENP-expressing cells were fixed 1 h or 4 h after release from nocodazole arrest or analyzed by time-lapse microscopy. (B) HeLa-S3 cells inducibly expressing 6Myc-tagged INCENP-WT or -S894A were fixed 1 h after release from nocodazole arrest, followed by immunostaining with anti–α-tubulin antibody (green) and Hoechst 33258 (blue). Phase-contrast images are shown in the right panels. Scale bar, 10 μm. (C) Percentage of cells undergoing multipolar division in HeLa-S3 cells inducibly expressing 6Myc-INCENP-WT, -S894A, or -S894D. Data represent the average of 30 telophase cells from three independent experiments. (D) HeLa-S3 cells inducibly expressing 6Myc-tagged INCENP-S894A were immunostained with anti–α-tubulin antibody (green) and Hoechst 33258 (blue). Phase-contrast images are shown in the right panels. Scale bar, 10 μm. (E) Percentage of cells with chromosome bridge (blue bars), lagging chromosomes (orange bars), or multipolar spindles (green bars) in HeLa-S3 cells inducibly expressing 6Myc-INCENP-WT, -S894A, or -S894D. Data represent the average of ∼50 mitotic cells from three independent experiments.