Apoptosis, cell cycle, and colony formation assays

Cells were cultured (3x10⁵/mL) for 6, 24, and/or 48 hours with UNC2025 or DMSO. Apoptotic and dead cells were detected by flow cytometry after staining with YO-PRO-1-iodide and propidium-iodide (7), cell cycle profiles were determined by assessment of propidium iodide staining in permeabilized cells using flow cytometry(17), and MTT reduction was determined as an indicator of viable cell number(17). Alternatively, ALL cell lines and patient samples were cultured in methylcellulose after treatment (10). AML cell lines were cultured in 0.35% Noble agar overlaid with medium containing UNC2025 or vehicle (15). Human mononuclear cells from normal bone marrow or umbilical cord blood were cultured in methylcellulose containing UNC2025 or DMSO (18). Colonies were counted after 7 (normal marrow) or 14 (umbilical cord blood, cell lines and patient samples) days.