Figure 5.
The phosphorylation of Cdt1 by the Cdk1 kinase impedes stable k-MT attachments and normal mitotic progression. (A) Histone H2B GFP-expressing, double thymidine synchronized, control HeLa cells (top panel) or those stably expressing RNAi-resistant WT (middle panel) or 2E3D mutant version (bottom panel) of Cdt1 were depleted of endogenous Cdt1 and the stability of K-fibers was assessed in each case by treatment with cold buffer 9 h after release from second thymidine arrest, as indicated. The cells were immunostained using anti-tubulin (green) and anti-Zwint1 (kinetochores, red) with the chromosomes counterstained using DAPI. (B) Same as in G, but the cells were fixed at both 9 and 10 h after release from second thymidine treatment followed by immunostaining using anti-tubulin (red) antibody and Histone H2B shown in green. (C) Quantification of cells in various stages of mitosis from B. Data presented as mean ± SD.

The phosphorylation of Cdt1 by the Cdk1 kinase impedes stable k-MT attachments and normal mitotic progression. (A) Histone H2B GFP-expressing, double thymidine synchronized, control HeLa cells (top panel) or those stably expressing RNAi-resistant WT (middle panel) or 2E3D mutant version (bottom panel) of Cdt1 were depleted of endogenous Cdt1 and the stability of K-fibers was assessed in each case by treatment with cold buffer 9 h after release from second thymidine arrest, as indicated. The cells were immunostained using anti-tubulin (green) and anti-Zwint1 (kinetochores, red) with the chromosomes counterstained using DAPI. (B) Same as in G, but the cells were fixed at both 9 and 10 h after release from second thymidine treatment followed by immunostaining using anti-tubulin (red) antibody and Histone H2B shown in green. (C) Quantification of cells in various stages of mitosis from B. Data presented as mean ± SD.

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