Fig. 3.
Aurora BK207R induces polyploidy and defective cell viability. (A) DNA content of HEK293 cells transiently transfected with a GFP empty vector (EV) and GFP-tagged vectors for wild-type Aurora B and Aurora B mutants (Aurora BK111M, Aurora BD205A and Aurora BK207R) and fixed 72 hours after transfection. HEK293 GFP-positive cells were detected and used for cell-cycle analysis. Overexpression of a SUMO-null Aurora-B protein (Aurora BK207R) deregulates cell-cycle progression inducing polyploidy in the same way as Aurora-BD205A kinase-dead form. (B) Bar graph quantification of cellular DNA content, showing a dramatic increase of polyploidy in cells overexpressing Aurora BD205A and Aurora BK207R mutants. (C) Immunofluorescence (GFP, green; DAPI, blue) images revealing that cells overexpressing Aurora BK207R are mostly multinucleated and display nuclear aberrancies. Scale bars: 10 μm. (D) Colony-formation assay in U2OS cells stably expressing GFP empty vector (EV) and GFP-tagged vectors for Aurora B (wild-type and mutants). U2OS GFP-positive cells were sorted 48 hours after transfection, replated in the presence of 1 mg/ml G418, then fixed 12 days later and stained with Crystal Violet to determine colony number. 50,000 GFP-positive cells were sorted and plated in 10 cm plates. (E) Quantification of the number of macroscopic colonies per 10 cm plate showing a significant (*P<0.001) reduction in the number of colonies in cells expressing Aurora BD205A and Aurora BK207R.