Phosphorylation of HOX11 During Mitosis in T-Cells is Associated with Aberrant Spindle Checkpoint Arrest and Reduced Early Apoptotic Cells in Cultures Expressing Wild-Type HOX11 but not the HOX11 T247E Mutant. (A) Immunoblot analysis of HOX11 and β-actin in ALL-SIL cells (upper panel) and Jurkat cells transiently transfected with HOX11 (lower panel). Cell lysates were prepared following treatment with aphidocolin (aph) to arrest cells in G1, aphidocolin followed by a 4 h release into fresh medium (aph+4) to arrest cells in S phase, genistein (gen) to arrest cells in G2/M and colchicines (colc), nocodazole (noc) or cytochalasin B (cytoB) to arrest cells in M. (A): asynchronous culture. (B) Mitotic index of Jurkat cells transiently transfected with empty vector, wild type HOX11 or the HOX11 T247E mutant then synchronized in mitosis with nocodazole. Nuclei were stained with Hoechst33342 (blue) and mitotic index determined by scoring condensed nuclei. Results are representative of three independent experiments. Error bars indicate the standard deviation. (C) Mitotic index of Jurkat cells transfected with wildtype and mutant HOX11. Nocodazole treated cells were harvested at 4-hour intervals and mitotic index determined. (D) Dysregulated HOX11 expression accelerates transit through the G2/M phase of the cell cycle. Jurkat cells were transfected with wildtype or mutant vectors. G1/S synchronized cells were released into the cell cycle then harvested at different time points and analyzed by flow cytometry for 4n DNA content by PI-staining. (E) One or four days post transfection, Jurkat cells expressing vector alone, HOX11-wt or HOX11-T247E cDNA were stained with Annexin V and PI and analyzed by flow cytometry to assess cell death. Percentages of early apoptotic cells are indicate. The results are representative of three independent experiments.