Inhibitors of cytokinesis do not block enucleation of mouse spleen erythroblasts. (A) Schematic showing the methodology of the short-term primary mouse spleen enucleation assay. Typically, between 12% and 18% of cells undergo enucleation in this short-term culture. (B) Results of enucleation assays performed in the presence of the cytokinesis inhibitors, blebbistatin, hesperadin, SU-6656, or nocodazole. The extent of enucleation was determined by quantifying the fractions of Syto 16-negative (permeable nucleic acid stain)/SytoX-negative (nonpermeable nucleic acid stain) cells by flow cytometry before and after a 5-hour incubation. The net percentage of enucleation was derived by taking the difference between the zero and 5-hour time points. Enucleation, compared with untreated, was calculated by multiplying 100 × ratio of the net percentage of enucleation of a drug-treated condition to the net percentage of enucleation of the corresponding solvent-control–treated condition. Cytochalasin D, a known inhibitor of enucleation, was included as a control for each experiment. Bars depict mean ± standard deviation of 3 independent experiments.