FIGURE 6:

Plk1 activity regulates phospho-S33/S37/T41 reactivity at spindle poles. (A) Pathways for regulation of centrosome separation by Plk1 at the onset of mitosis. (B, D) U2OS cells synchronized in mitosis by double-thymidine block and release were treated with control (2% DMSO), Plk1 inhibitor (100 nM BI2536), Eg5 inhibitor (100 μM monastrol), or GSK3 inhibitor (20 μM SB21673) for the last 9 h of the second release, stained for DNA with DAPI, and immunostained with antibodies as indicated. For presentation of control spindles and different treatments, images were taken at identical exposure times and identically contrast enhanced for each stain. Scale bar, 10 μm. (C, E) Graphs show levels of β-catenin (C) and phospho-S33/S37/T41 immunofluorescence (E; AU, arbitrary units) at spindle poles of untreated U2OS cells or cells treated as described in B and D. Error bars, SEM of ≥16 spindle poles for C and SEM of ≥10 spindle poles for E; ***p < 0.001. Original unmodified images taken at identical exposure times were measured for controls and different treatments. The data are representative of two independent experiments done with all cell lines under identical conditions.