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Fig. 2. Depletion of Bub3 in Drosophila S2 cells results in slower progression through prophase and premature exit from mitosis. (A) Western blot showing depletion of Bub3 after RNAi at different times. 
-tubulin antibody was used as loading control. (B) Immunolocalization of Bub3 in control and Bub3 dsRNA-treated cells, after 96 hours in culture. Polo was used to label kinetochores. Merged images are shown with DNA in blue, polo in red and bub3 in green. (C) Mitotic index of control and S2 cells treated with Bub3 dsRNA for 96 hours after incubation in colchicine for different times. Mitotic index was determined as the number of phospho-histone H3 (PH3)-positive cells over the total cell population. (D) Percentage of mitotic cells showing sister chromatid separation (SCS, anaphases plus PSCS) after incubation with colchicine, in control and Bub3-depleted cells. (E) RNAi-treated Bub3 cells showing SCS after incubation with colchicine. DNA is shown in red and PH3 in green. (F) Proliferation rate of control and Bub3 RNAi-treated cells. (G) Time course analysis of the mitotic index after Bub3 RNAi, assessed by PH3 staining. (H-J) Analysis of mitotic progression of control and S2 cells treated with RNAi. Mitotic cells were identified by immunostaining with PH3 specific antibodies. (H) Quantification of cells in anaphase and prometaphase-like cells in which sister chromatids are clearly separated over time. (I) Treatment of S2 cells with Bub3 RNAi reduces the number of cells in prometaphase and metaphase over time. (J) Depletion of Bub3 causes a marked increase in the number of cells in prophase by 72 hours after treatment. Bar, 5 µm.
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