Figure 2.
The Ska1 complex is essential for the recruitment of Cdt1 to the kinetochores and spindle microtubules. (A) HeLa cells treated with either scramble control (siControl) or siRNA against endogenous SKA3 (siSka3) were fixed using paraformaldehyde. Representative images of cells immunostained with antibodies against Ndc80/Hec1 (a kinetochore marker) in far red, SKA3 in green, and Cdt1 in red are shown. Scale bar, 5 μm. (B) A bar graph showing the quantification of kinetochore staining intensity after siRNA treatment in each case (n = 110 kinetochores, >5 cells). Data presented as mean ± SD, two-tailed t test, *** = P < 0.0001. (C) HeLa cells treated with either siControl or siSKA3 were fixed using methanol. Representative images of cells immunostained with antibodies against tubulin in green and Cdt1 in red are shown; DAPI-stained chromosomes are shown in blue in the merged image. Scale bar, 5 μm. (D) A bar graph showing the quantification of Cdt1 spindle microtubule staining intensity after siSKA3 treatment as compared to siControl (n = 13 cells). Data presented as mean ± SD, two-tailed t test, *** = P < 0.0001. (E–G) HeLa cells stably expressing either GFP-SKA1 full-length (E and F) or GFP-SKA1ΔMTBD (G) were treated with siSKA3 to knockdown the endogenous SKA3 and the expression of siRNA-resistant GFP-SKA1 or GFP-SKA1ΔMTBD proteins was induced by adding 2.5 µg/ml of Doxycycline (+Dox) for 48 h. Control cells were treated similarly with the siSKA3 in each case, but no doxycycline was added (−Dox). The cells were then fixed and immunostained with antibodies against GFP for SKA1-FL or SKA1 ΔMTBD (second column in E and G), Cdt1 (third column and in red in the merge images), and microtubules (fourth column and in blue in the merge images) with the chromosomes counterstained using DAPI (first column). Scale bar, 5 μm. (F) A bar graph showing the quantification of Cdt1 spindle microtubule staining intensity from E in the presence and absence of Doxycline induction (n = 22 cells). Data presented as mean ± SD, two-tailed t test, *** = P < 0.0001.

The Ska1 complex is essential for the recruitment of Cdt1 to the kinetochores and spindle microtubules. (A) HeLa cells treated with either scramble control (siControl) or siRNA against endogenous SKA3 (siSka3) were fixed using paraformaldehyde. Representative images of cells immunostained with antibodies against Ndc80/Hec1 (a kinetochore marker) in far red, SKA3 in green, and Cdt1 in red are shown. Scale bar, 5 μm. (B) A bar graph showing the quantification of kinetochore staining intensity after siRNA treatment in each case (n = 110 kinetochores, >5 cells). Data presented as mean ± SD, two-tailed t test, *** = P < 0.0001. (C) HeLa cells treated with either siControl or siSKA3 were fixed using methanol. Representative images of cells immunostained with antibodies against tubulin in green and Cdt1 in red are shown; DAPI-stained chromosomes are shown in blue in the merged image. Scale bar, 5 μm. (D) A bar graph showing the quantification of Cdt1 spindle microtubule staining intensity after siSKA3 treatment as compared to siControl (n = 13 cells). Data presented as mean ± SD, two-tailed t test, *** = P < 0.0001. (E–G) HeLa cells stably expressing either GFP-SKA1 full-length (E and F) or GFP-SKA1ΔMTBD (G) were treated with siSKA3 to knockdown the endogenous SKA3 and the expression of siRNA-resistant GFP-SKA1 or GFP-SKA1ΔMTBD proteins was induced by adding 2.5 µg/ml of Doxycycline (+Dox) for 48 h. Control cells were treated similarly with the siSKA3 in each case, but no doxycycline was added (−Dox). The cells were then fixed and immunostained with antibodies against GFP for SKA1-FL or SKA1 ΔMTBD (second column in E and G), Cdt1 (third column and in red in the merge images), and microtubules (fourth column and in blue in the merge images) with the chromosomes counterstained using DAPI (first column). Scale bar, 5 μm. (F) A bar graph showing the quantification of Cdt1 spindle microtubule staining intensity from E in the presence and absence of Doxycline induction (n = 22 cells). Data presented as mean ± SD, two-tailed t test, *** = P < 0.0001.

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