CA-WASp mitotic abnormalities are sensitive to the Aurora B pathway and involve increased kinetochore microtubule intensity. (A) Western blot showing Aurora B-BFP overexpression in HT1080 and U937 cells. (B) Percentage binucleated and micronucleated U937 and HT1080 cells after 4-day expression of GFP-CA-WASp in cells overexpressing Aurora B or inhibition of Aurora B with 5nM AZD1152 for 48 hours. Images show examples of binucleated and micronucleated HT1080 cells. (C) Confocal images of HT1080 cells blocked in metaphase by proteasome inhibition followed by 10 minutes in ice-cold media stained for α-tubulin (red) and Hec1 (green, a kinetochore protein) to show kinetochore microtubule fibers. Bar = 5 μm (top panels) and 1 μm (bottom panels). (D) Quantification of kMT intensity from cells prepared as in panel C. Values are relative to the mean control value from each experimental repeat (n ≥ 3). kMT intensity was measured from the fluorescence intensity of the entire spindle (top panels, white dashed areas) and also as an average value of 0.25-μm² sections of kMT fibers at 10 individual kinetochores per cell (bottom panels, white dashed areas). Each point on the chart represents the kMT intensity of an individual cell, with the values from the cells shown in panel C highlighted as red points. Confocal microscopy performed with a Zeiss LSM 710 inverted confocal microscope with a 63× P-Apochromat NA 1.4 oil-immersion objective, with fluorochromes eGFP-WASp, Alexa Fluor 568, and Alexa Fluor 647.