Figure 3.
A dose-dependent increase in mitotic arrest with chromosome congression defects is caused upon treatment with Compound 9 and its enantiomers. (A) Cells were treated with Compound 9 and its enantiomers, Compounds (-)-9 and (+)-9 (at 200, 100, 50, 25, 12.5, 6.25 and 0 μM) and corresponding DMSO controls for 12h. Mitotic cells scored as phospho-histone H3-stained cells per 2000 Hoechst 33342-stained nuclei in a high-content screening platform as described earlier.27 Each bar is a mean of three replicates ± S.E.M. The data presented is representative of two independent experiments. (B) HeLa cells were treated with compound (-)-9 / DMSO for 12 h as shown in Fig. 3A. The cells were fixed and stained for DNA and β-tubulin. Mitotic cells were identified by microscopy and scored under three. categories, (a) misaligned chromosomes and monopolar spindles; (b) misaligned chromosomes and bipolar spindles and (c) aligned chromosomes and bipolar spindles. The quantification of cellular phenotype is shown in the histogram. (C) Representative maximal-intensity projection images of cells in each category (as in Fig. 3B) showing DNA in blue and spindle microtubules in red. Scale bar is 3 μm.