Fig. 6.

Aurora B^K207R does not rescue mitotic defects in Aurora-B-knockout MEFs. (A) Schematic representation of the protocol followed for rescue upon acute deletion of Aurora B in quiescent cells. Primary (passage 2) Aurkb(lox/lox) MEFs were seeded at 80% confluence and infected the following day with retroviruses expressing Aurora-B^WT or Aurora-B^K207R. Next day, cultures reach confluence and were cultured in 0.1% FBS and infected with adenoviruses expressing Cre recombinase (AdCre). Two days after the infection, cells were split in new plates at low confluence in the presence of 10% FBS to induce entry into the cell cycle. Mitotic cells were analyzed by immunofluorescence during the first round of division (28 hours after splitting). (B) Acute deletion of Aurora B in MEFs provokes chromosome misalignment and spindle defects that are rescued by exogenous, properly localized, wild-type Aurora B [Aurkb(Δ/Δ); Aurora B^WT; green signal in merged images]. The exogenous Aurora B^K207R, however, remains diffuse on chromosome arms and it does not rescue the Aurkb-null defects resulting in chromosome misalignment and impaired cytokinesis in Aurkb(Δ/Δ); Aurora B^K207R cells. DNA (DAPI; blue) is delineated in the insets. α-tubulin is in red in merged images. Scale bars: 10 μm.