FIGURE 2.

Phosphorylation of Sds22 is critical for mitotic progression. A, the expression levels of Sds22^WT-GFP, Sds22^5A-GFP, and Sds22^5D-GFP in HeLa cells co-transfected with mCherry-H2B. Cells were collected at 48 h after transfection and analyzed by Western blots using indicated antibodies. Tubulin served as loading control. B, live cell imaging of chromosome movements in HeLa cells expressing wild type and mutant Sds22. Aliquots of HeLa cells were transiently transfected to express various GFP-tagged Sds22 plasmids with mCherry-H2B. 36 h after transfection; images were acquired at the indicated time points. Note that expression of Sds22^5A-GFP resulted in mitotic arrest phenotype, and expression of Sds22^5D-GFP resulted in mitotic delay phenotype. Scale bar, 10 μm. C, statistical analyses of mitotic phenotypes as in B. Error bars represent means ± S.E.; >20 cells of each category from three independent preparations. Student's t test was used to calculate p value for comparison. **, p < 0.01. D, the expression levels of Sds22^WT-GFP and its mutants in endogenous Sds22-depleted HeLa cells. Cells were collected at 48 h after transfection and analyzed by Western blots using indicated antibodies. Tubulin served as loading control. E, live cell imaging of chromosome movements in Sds22 depleted HeLa cells expressing wild type and mutant Sds22 proteins. Note that in Sds22-depleted HeLa cells, expression of Sds22^5A-GFP resulted in mitotic arrest phenotype, and expression of Sds22^5D-GFP resulted in mitotic delay phenotype. Scale bar, 10 μm. F, statistical analyses of mitotic phenotypes seen in live cell imaging as in E. Error bars represent means ± S.E.; >20 cells of each category from three independent preparations. Student's t test was used to calculate p value for comparison. ***, p < 0.001.