Fig. 4. Bub3-depleted cells exhibit lower levels of cyclin B during G2 and mitosis. (A) Analysis of cyclin B at different stages of the cell cycle in control and Bub3 RNAi-treated cells for 96 hours, followed by incubation for 6 hours in colchicine. -tubulin staining (red) was used to discriminate between G1/S, G2 and mitotic cells. DNA is shown in white and cyclin B in green. Images show that Bub3-depleted cells do not accumulate cyclin B to control levels in G2 or during mitosis. (B) Quantification of cyclin B levels. The mean pixel intensity per cell in control and RNAi-treated cells was measured at different cell cycle stages (see also supplementary material Table S2). ***P<0.0005 when compared to intensity in control cells. (C) Cyclin B accumulation during G1/S and G2 in control and Bub3-depleted cells after 96 hours in culture without colchicine treatment. -tubulin staining (red) was used to classify cells as before, cyclin B is shown in green and DNA in white. Bub3-depleted cells fail to accumulate cyclin B during G2, unlike control cells. (D) Quantification of cyclin B levels (see also supplementary material Table S3). ***P<0.0005 when compared to intensity in control cells. (E) Analysis of cyclin B levels by western blotting of total protein extracts (10 µg) isolated from control or RNAi-treated cells before or after 3 and 6 hour incubations with colchicine. -tubulin was detected as a loading control. (F) Quantitative analysis of the western blot shown in E. Bar, 5 µm.