Fig. 5. Mutation of the APC/C subunit cdc27 in bub3¹ mutant cells allows cyclin B accumulation during G2 and restores normal progression through early mitotic stages. (A) Neuroblasts of different genotypes were isolated, incubated in colchicine (1 hour), fixed and stained for cyclin B (green). DNA was counterstained with DAPI (white). Images show high levels of cyclin B in cdc27 mutant cells whereas cdc27;bub3¹ double mutant cells show normal cyclin B levels during G2. (B) Quantification of cyclin B levels. The mean pixel intensity per cell in the wild type and mutant strains at different cell cycle stages was measured (see also supplementary material Table S4). *P<0.05; **P<0.005; ***P<0.0005 when compared to intensity in corresponding control cells. (C) Mitotic index of wild-type (control), double mutant (cdc27;bub3¹) and single mutant (cdc27 or bub3¹) cells. Mutation of cdc27 in a bub3¹ background leads to an increase in the mitotic index relative to bub3¹ alone, but still reduced when compared to the wild-type index. (D) Quantification of cells in prophase for the different genetic backgrounds showing that double mutant cells (cdc27; bub3¹) transit normally through prophase. (E) A non-degradable form of cyclin B (CyCBND) was expressed in bub3¹ mutant neuroblasts (Mz1061;CycBND-GFP;bub3¹). Mz1061 was used to drive neuroblast expression. Mz1061;CycBND-GFP neuroblasts were used as a control. Expression of a non-degradable form of cyclin B in bub3¹ neuroblasts restores the mitotic index to wild-type values. Bar, 5 µm.