Figure 2

Wiskostatin inhibits cytokinesis but not mitosis. A. HeLa cells were synchronised in prometaphase with nocodazole and treated with vehicle or indicated concentrations of wiskostatin. Cells were harvested at indicated time points and DNA content determined by FACS analysis. A similar scale was used for each histogram. This result is representative of at least 3 independent experiments. B. HeLa cells synchronised as previously described were treated with vehicle (-) or 10 μM wiskostatin (+) and harvested at indicated time points. Cyclin B1, phosphorylation of Ser 10 of histone H3 and cofilin levels were determined by immunoblot analysis with specific antibodies. This result is representative of at least 5 independent experiments. C. HeLa cells stably expressing GFP-tagged histone H2B were synchronised as previously described, treated with vehicle (Control) or 10 μM wiskostatin (Wiskostatin 10 μM) and fixed after 300 minutes. F-actin was stained and nuclei per cell determined. Percentage of binucleated cells was then calculated. 536 cells treated with vehicle and 569 cells treated with wiskostatin were count from three independent experiments. Error bars represent standard error of the mean (SEM). D. Assynchronous HeLa cells stably expressing GFP-tagged histone H2B were treated with vehicle (Control) or 5 μM wiskostastin (Wiskostatin 5 μM) for 24 hours and were then fixed. Percentages of binucleated cells were determined as previously indicated. 629 cells treated with vehicle and 640 cells treated with wiskostatin were count from three independent experiments. Error bars represent SEM. Representative images from time lapse movies of HeLa cells stably expressing GFP-tagged histone H2B synchronised as previously described, treated with vehicle (E) or 10 μM wiskostatin (F). Images represent merged between DIC and fluorescent (Histone H2B) images. Time indicated as hours: minutes.