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. 2015 Mar 18;14(10):1568–1582. doi: 10.1080/15384101.2015.1026489

Figure 6.

Figure 6.

Phosphorylation of CHO1 at S716S717 is required for cytokinesis. (A) The percentages of multinucleated cells in HeLa-S3 populations stably expressing 6Myc-vector or 6Myc-CHO1-WT (wild type) that were transfected with a control (GL2) or CHO1/MKLP1-specific siRNA. The cells were fixed 72 h after transfection. (B) Representative images of immunostained multinucleated cells (including binucleated cells). (C) The percentages of multinucleated cells in a HeLa-S3 population stably expressing 6Myc-tagged vector alone or CHO1-WT, -SA, -SD, -2SA, or -3SA. (D) The percentages of multinucleated cells in Lats1-KO mouse embryo fibroblasts (MEFs) transfected with 6Myc-tagged vector alone, large tumor suppressor (Lats)1-WT, or CHO1-WT. The cells were fixed 72 h after transfection. (E) The percentages of HeLa-S3 cells stably expressing 6Myc-tagged vector alone, CHO1-WT, -SA, or -SD at each stage of the cell cycle. The cells were synchronized by a thymidine single block-and-release (10 h). (F) The centrosomal localization of LIM-kinase 1 (LIMK1)-pT508 in G2 phase HeLa-S3 cells stably expressing 6Myc-tagged vector alone or CHO1-WT, -SA, or -SD. The cells were synchronized by a thymidine single block-and-release (10 h). Scale bar, 10 μm. The bar graphs show the intensities of the LIMK1-pT508 (center) and γ-tubulin (right) signals on the centrosomes in the G2 phase. Data represent the mean ± SD of n = 3 independent experiments (30 cells per experiment). (G) Immunoblot analyses of Cofilin-pS3 in HeLa-S3 cells transiently expressing 6Myc-tagged vector alone or CHO1-WT, -SA, or -SD. Transfected HeLa-S3 cells were treated with nocodazole (80 ng/ml) for 16 h prior to releasing for 2 h with (+) or without (−) MG132 (5 nM). (H) Midbody localization (arrow) of ectopic 6Myc-CHO1-SA in HeLa-S3 cells undergoing cytokinesis. The asterisks indicate mislocalization of CHO1-SA. Scale bar, 10 μm. The bar graph shows the percentages of cells expressing 6Myc-tagged vector alone or CHO1-WT, -SA or -SD with midbody localization of the exogenous protein during late telophase. (I) Midbody localization of Ect2 during cytokinesis in HeLa-S3 cells stably expressing 6Myc-tagged vector alone or CHO1-WT, -SA, or -SD. The arrow indicates mislocalization of Ect2. Scale bar, 10 μm. The bar graph shows the percentages of cells undergoing cytokinesis with midbody Ect2 signals. (B–E, H, I) Data represent the mean ± SD of n = 3 experiments (more than 100 cells per experiment).