Figure 4: Aneuploid brains present an extended G1 phase.

(a) Pictures of WT (upper panel), SakOE,mad2 (middle panel) and bub3 (lower panel) brains stained for EdU (5-ethynyl-2’-deoxyuridine) (second panel, shown in red in the merged panel), PH3 (third panel, shown in green in the merged panel) and Elav (right panel) labelling cells in S-phase, in M phase and neurons respectively. The CB is marked by the yellow dashed line. Scale bar, 50 μm. (b) Graph bar showing the quantification of cell cycle phase distribution in WT (4830 cells from eight lobes) SakOE,mad2 (3220 cells from 10 lobes) and bub3 (2973 cells from eight lobes) brains. Among the non-Elav cells, percentages of each cell cycle phases were estimated: EdU⁺: cells in S phase, EdU⁺PH3⁺ or PH3⁺: cells in G2/M Edu⁻,PH3⁻: cells in G1. In the CB, aneuploid brains present a higher percentage of cells in G1. Unpaired t-test. ***(P<0.001). (c) Diagram of the fluorescence ubiquitination cell cycle indicator (FUCCI) system. Fusions to GFP-E2F and RFP- Cyclin B are used as indicators of cell cycle progression⁵⁰. G1 cells show nuclear green signal, S-phase cells show red cytoplasmic signal, and G2/M cells show green and red signals in the nucleus and cytoplasm respectively. M-phase cells round up and appear yellow. (d) Pictures of WT (top) and SakOE,mad2 (bottom) brain lobes expressing the FUCCI system. The insets show G1 cells. In SakOE,mad2, the large cell with three nuclei probably resulted from cytokinesis defects described in Fig. 1. This type of cell is never seen in WT. Scale bar, 50 μm. (e) Graph bar showing the quantification of cell cycle phase distribution in WT (280 cells from 12 lobes) SakOE,mad2 (261 cells from 18 lobes) brains. Unpaired t-test. **(P=0.0092).