Fig. 2.
Aurora-AG198N prevents chromosome misalignment and cell premature exit from mitosis caused by Aurora-B knockdown. (A) Immunofluorescence photomicrographs of HeLa cells cotransfected with Aurora-B RNAi and RFP-H2B (red) at a ratio of 40:1 and labeled with Aurora-B antibody (green). Note that those cells expressing RFP-H2B showed disperse staining of Aurora-B with decreased fluorescence intensity, as indicated by arrows. Thus, in addition to chromosome morphology, RFP-H2B was a marker for Aurora-B knockdown. DNA labeled with DAPI (blue). (B) HeLa cells induced by tetracycline to express Aurora-AWT or Aurora-AG198N were cotransfected with Aurora-B RNAi and RFP-H2B at a ratio of 40:1, and subjected to time-course fluorescence microscopy. Chromosome morphology, visualized by RFP-H2B staining, was filmed every 5 min. With Aurora-B knockdown, cells could not properly align the chromosomes and exited mitosis without chromosome separation and cytokinesis. Induced expression of Aurora-AG198N, but not Aurora-AWT, prevented this abnormality. Representative cells can be viewed as movies or still images in Movie S1, Movie S2, Movie S3, Movie S4, Movie S5, Movie S6, Movie S7, and Movie S8. (C) Quantitative characterization of mitotic HeLa cells treated as in B. Dark-gray bars represent the percentage of cells from prophase to metaphase, and light-gray bars represent the percentage in anaphase. Cells were classified by chromosome morphology visualized by RFP-H2B. Each data point represents 3 independent experiments, each measuring 250 mitotic cells, and error bars indicate SD (P < 0.01).