Figure 4.

Overexpression of cyclin E2 promotes polyploidy in association with Cdt1 downregulation. (A) Schematic of nocodazole-mediated arrest in T-47D cells constitutively expressing cyclins E1 and E2: cells are arrested at G₁ with thymidine, released into the cell cycle and then arrested at mitosis with nocodazole. (B) Quantitation of polyploid cells after nocodazole arrest by flow cytometry of propidium iodide-stained cells gated for >4N content. N = sets of chromosomes. Data were analysed by one-way ANOVA, with Tukey’s multiple comparisons test. (C) Representative examples are shown of each cell line after nocodozole arrest, where they are stained with propidium iodide and analysed by flow cytometry to detect >4N cells. N = sets of chromosomes. (D) Western blotting for cyclin E1, cyclin E2, Cdt1 and GAPDH of cell lysates from cells synchronised with thymidine, and then arrested for 24 h with nocodazole. The uncropped western blot figure in Figure S4. (E) Quantitation of Cdt1 expression by densitometry of duplicate experiments, error bars indicate range. Data were analysed by one-way ANOVA, with Tukey’s multiple comparisons test.