erformed with a 488 diode laser on one KT pair per cell. Overall bleaching was corrected using the signal intensities at a cytoplasmic region not targeted for photobleaching. Fluorescence recovery half-times and plateaus were determined by non-linear curve fitting based on a one-phase association in Prism software. Colony formation assay Asynchronous cell cultures were plated on 6-well plates. After 7 days of proliferation in the presence of the indicated drugs, the cells were fixed with ice-cold methanol at -20C and stained with 0.1% Cresyl Violet according to standard procedures. Dried culture plates were scanned and intensities measured using ImageJ after black-and-white-conversion and inversion of the images. IC50 isobolograms BAY-320 plus Paclitaxel combination studies were conducted with HeLa and NCI-H1299 cells. Cells were plated into 384-well plates at 600 or 700 cells per well. After 24 hr, cells were treated with BAY-320 and Paclitaxel for single compound treatments, and in nine different fixed-ratio combinations of BAY-320 and Paclitaxel . Cell viability was assessed after 96 hr exposure, using the Cell Titre-Glo Salvianic acid A price Luminescent Cell Viability Assay. IC50 values were determined by means of a 4-parameter fit after normalization of measurement data to vehicle -treated cells and readings taken immediately before compound addition. IC50 isobolograms were plotted with the actual concentrations of the two compounds on the x- and y-axis, and the combination index was calculated according to the median-effect model of Chou-Talalay. A CI of 0.8 was defined as more than additive interaction, and a CI of 1.2 was defined as antagonistic interaction. rAAV-mediated gene targeting For gene targeting, homology arms to human Bub1 gene were amplified from RPE1 cell genomic DNA. Targeting constructs allowing the insertion of an EGFP tag C-terminal to Bub1 were assembled by 4-piece ligation in a NotI-digested pAAV vector. Recombinant adenovirus-associated virus particles were generated as previously PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19826300 described. RPE1 cells were infected with 3 ml of viral supernatant for 48 hr and then expanded into fresh medium for an additional 48 hr. FACS sorting was used to select EGFP-positive cells, as previously described. To facilitate detection of fluorescence at mitotic stages, cells were synchronized with RO-3306 for 18 hr and released into nocodazole for 2 hr, before they were trypsinized and subjected to sorting in the continued presence of nocodazole. Infected or uninfected cells were filtered and EGFP-positive cells were isolated on an Aria IIIu cell sorter by selecting the 514/30 channel against a Baron et al. eLife 2016;5:e12187. DOI: 10.7554/eLife.12187 20 of 26 Research article Cell biology 585/42 filter detecting cellular autofluorescence. Single cells were sorted into 96-well plates filled with conditioned medium and positive clones screened for by fluorescence microscopy. Acknowledgements We thank the project team at Bayer Pharma AG for their contribution. We also thank Prof. GJPL Kops and Dr. M Vleugel for providing the LAP-Bub1 
and LAP-Bub1 plasmids, Stephen Taylor for the RPE1 H2B-GFP cell line, Zhen Dou for the generation of the MCAK antibody and Michael Lampson as well as Daniel Gerlich for Aurora B biosensor constructs and HeLa Kyoto cell lines. We also thank the Biozentrum’s Imaging Core Facility for support and Janine Zankl for help with FACS analysis. APB was funded by a fellowship from the `Fellowships for Excellence’ International PhD
