AZ0108

EC50s were measured in a high-content phenotypic assay that was also used to screen for small molecules that block centrosome clustering and cause a multi-polar spindle phenotype. This cell-based centrosome declustering assay begins with treatment of HeLa cells with increasing concentrations of compound. After 24 h, the cells are fixed and then the mitotic cells are identified by treatment with a cyclin B antibody. Next, the cells are treated with an antibody towards pericentrin to label the centrosomes. After that, an automated process generates an overlay of the two images and analyzes it to calculate the number of centrosomes observed in each mitotic cell. Mitotic cells with greater than four centrosomes are then assumed to have a multi-polar spindle phenotype Finally, a declustering EC50 is calculated from a doseresponse curve generated from a plot of the percentage of mitotic cells with a multi-polar spindle phenotype at eight concentrations ranging from 3 nM to 10 uM.