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Files in this Data Supplement:
Fig. S1. bub31 mutant neuroblasts fail to accumulate wild-type levels of cyclin B during G2 and mitosis. (A) Wild-type and bub31 mutant neuroblasts were isolated, incubated in colchicine and fixed. Cells with low levels of cyclin B and no chromosome condensation were classified as G1/S, those with higher levels of cyclin B and no chromosome condensation as G2 and those with visible chromosome condensation as mitotic. Cyclin B is shown in green and DNA in white. The results show that mutant cells are unable to accumulate normal levels of cyclin B at G2 or during mitosis. (B) Quantification of cyclin B levels by measuring the mean pixel intensity per cell in wild-type and mutant cells at different cell cycle stages. (C) Western blot of total protein extracts from wild-type and bub31 mutant neuroblasts, before or after treatment with colchicine, show that mutant cells never accumulate wild-type levels of cyclin B. a-tubulin was detected as a loading control. (D) Graphic representation of the variation of cyclin B levels shown in C. Control cells without colchicine treatment were used as standard cyclin B levels. All values were normalized relative to a-tubulin levels. Bar, 5 mm.
Movie S1. Live imaging of an S2 Drosophila control tissue culture cell during mitosis. The cells are stably transformed with a construct to express GFP-a-tubulin imaged from start of prophase as shown by the presence of separating spindle poles until mitotic exit as shown by the telophase configuration of spindle microtubules. Time is shown as minus before NEBD and as plus after NEBD.
Movie S2. Live imaging of an S2 Drosophila tissue culture cell treated with bub3 dsRNA for 96 hours before following its progression through mitosis. The cells are stably transformed with a construct to express GFP-a-tubulin imaged from start of prophase as shown by the presence of separating spindle poles until mitotic exit as shown by the telophase configuration of spindle microtubules. Time is shown as minus before NEBD and as plus after NEBD.
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