EM011 induces apoptosis. Panels A-D show flow cytometric analysis of phosphatidylserine (PS) exposure in untreated CEM (A), untreated CEM/VLB100 (B), EM011-treated CEM (C), and EM011-treated CEM/VLB100 (D) cells. Alexa-Fluor 488 conjugate of annexin V was used in combination with PI to distinguish among 3 subpopulations: PI^- and Alexa-Fluor 488^- population indicates viable cells (bottom left quadrant); PI^- and Alexa-Fluor 488⁺ population indicates early apoptotic cells (lower right quadrant); PI⁺ and Alexa-Fluor 488⁺ population indicates late apoptotic cells (top right quadrant). Representative annexin V-stained cells are shown in panel F (EM011-treated) as green rings surrounding early apoptotic cells (solid arrowhead) as opposed to the control untreated cells in panel E, where green rings are absent. However, some nonspecific staining is evident. Scale bar = 20 μm. (G-J) EM011 reduces mitochondrial transmembrane potential in a time-dependent manner. CEM/VLB100 cells were treated with EM011 for 0, 24, 48, and 72 hours, incubated with 50 nM DiOC6, and then analyzed by flow cytometry. The x-axis represents the DiOC6 fluorescence intensity, and the y-axis represents the number of cells. (K) Quantitation of caspase-3 activity in EM011-treated CEM and CEM/VLB100 cells. Cells were treated with 10 μM EM011 for 0, 24, 48, and 72 hours, and caspase-3 activity was analyzed using the luminogenic substrate Z-DEVD-aminoluciferin. ^*P < .01. (L) Representative immunoblot of cleaved caspase-3 and cleaved PARP along with the loading control, actin. Results are representative of 3 independent experiments performed.