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

Fig. 14.

Fig. 14

Phosphorylation of INCENP at Ser894 is required for the completion of multipolar cell division during cytokinesis. (A) HeLa-S3 cells inducibly expressing 6Myc-INCENP-WT or -S894A were synchronized as in Fig. 13A. Representative images show differential interference contrast images captured every 12 min by time-lapse analysis. Arrows show that INCENP-WT cells completed cytokinesis and INCENP-S894A cells failed to complete cytokinesis. (B) INCENP siRNA complementation assay. HeLa-S3 cells inducibly expressing 6Myc-INCENP-WT, -S894A, -S894D, or vector alone were treated with 2 μg/ml Dox for 6 h before siRNA transfection. Cells were transfected with siRNAs against the 3′UTR of INCENP (siINCENP) or the firefly luciferase gene (GL2, a negative control). After transfection, cells were cultured for 48 h in the presence of 2 μg/ml Dox. Cells were fixed and stained with Hoechst 33258, followed by counting of cells and nuclei by microscopy. The color bar graphs show the percentage of cells with the indicated number of nuclei. Data represent the average of ∼50 cells from three independent experiments. (C) HeLa-S3 cells inducibly expressing 6Myc-tagged INCENP-WT or -S894A were transfected with siINCENP and fixed 4 h after release from nocodazole arrest, followed by co-immunostaining with anti-Myc (red) and anti–α-tubulin antibodies (green). DNA was stained with Hoechst 33258 (gray or blue). Scale bar, 50 μm. (D) Percentage of cells with the indicated number of nuclei in HeLa-S3 cells inducibly expressing 6Myc-INCENP-WT, -S894A, -S894D, or vector alone under the same conditions as in C. In Myc-positive cells, cells and nuclei were counted by microscopy. Orange bars show the proportions of bi- and multinucleated cells. Data represent the average of ∼90 cells from three independent experiments. (E) HeLa-S3 cells inducibly expressing 6Myc-tagged INCENP-WT or -S894A were transfected with siINCENP and fixed 4 h after release from nocodazole arrest. Percentage of cells with multiple nuclei (more than two nuclei) in HeLa-S3 cells inducibly expressing 6Myc-INCENP-WT, -S894A, -S894D, or vector alone. Data represent the average of ∼90 cells from three independent experiments.