Fig. 3. In vivo analysis of S2 cells shows that Bub3-depleted cells transit faster through mitosis, despite a delay in prophase. (A) Selected images from time-lapse movies (see supplementary material Movie 1) of either control or Bub3 RNAi-treated cells expressing GFP-tubulin recorded every 2 minutes from the time asters first appeared to the re-formation of the daughter nuclei. The two movies have been aligned setting nuclear envelope breakdown (NEBD) as 0. In control cells, NEBD occurred approximately 17 minutes after asters were clearly visualized, whereas anaphase started 32 minutes after NEBD. In Bub3 RNAi-treated cells, NEBD occurred 32 minutes after aster formation, showing a significant delay in prophase. Anaphase started approximately 20 minutes after NEBD, indicating a rapid transition from prometaphase to anaphase. (B) Quantitative analysis of the different cell cycle stages, in both control (n=14) and Bub3 RNAi-treated cells (n=29). **P<0.005 when compared to corresponding stages in control cells. (C) Analysis of prophase cells by PH3 labelling reveals that the majority of the Bub3-depleted cells (53%) exhibits strong PH3 labelling and DNA condensation but no aster formation, in contrast to the control population where PH3 staining correlated with aster formation in more than 98% of cells (n=40).