HeLa cells were transfected with control or indicated siRNA (ON-TARGETplus SMARTpool siRNA, Dharmacon) for 48 h, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100/PBS, and stained with 0.5 μg/ml Hoechst 33342, rat anti-α-tubulin (Serotec, Oxford, United Kingdom) and either rabbit anti-Cdc27, Eg5, or cyclin B or mouse anti-centrin. For acute OA treatment, cells were treated with 175 nM OA for 13 min before fixation and staining. Slides were mounted with ProLong Gold anti-fade reagent (Invitrogen), and projection images (10-μm stacks captured every 0.5 μm) were captured with a Zeiss Axio Imager.Z1 microscope (Thornwood, NY) equipped with a CoolSNAP HQ camera (Photometrics, Tucson, AZ) and operated with SlideBook 4.2 (Intelligent Imaging, Denver, CO) at 63× (NA 1.4) at room temperature. One hundred cells were analyzed to determine the percentage of cells with Cdc27 spindle pole localization in control, indicated siRNA, and acute OA-treated cells. A 2 × 2-μm square was drawn around each of 20 spindle poles from control, indicated siRNA, or acute OA-treated cells, and the mean fluorescence intensity of Cdc27 or cyclin B spindle pole staining was plotted as arbitrary units (AU). Additionally, intensity measurements were taken along an axis intersecting the two spindle poles and the fluorescence intensity was graphed as arbitrary units (AU). Image processing was performed using Adobe Photoshop CS2 (version 9.0.2; San Jose, CA).