TDZD-8 induces cell death with extremely rapid cell death kinetics showing loss of membrane integrity. (A) Percent viability assessed at the indicated time points for CD34⁺CD38⁻ populations of primary AML specimens (n = 8) treated with TDZD-8 (■) or PTL (□). Percent viability is represented relative to untreated control. (B) Percent viability assessed at the indicated time points for unfractionated primary AML specimens (n = 17) treated with TDZD-8. Percent viability is shown relative to untreated controls. Error bars represent SEM. (C) Cells were treated with 20 μM TDZD-8 for the indicated periods of time, then washed and placed in culture until analysis at 24 hours. Percent viability represented relative to untreated control. (D) Percentage of CFUs relative to untreated control. Cells were washed and placed in methylcellulose culture medium at the indicated time points after the addition of TDZD-8. (E) Loss of membrane integrity assessed by YoPro-1 uptake after 15 minutes of TDZD-8 treatment. Multispectral imaging flow cytometry shows the internalization of YoPro-1. Cells were stained with CD45 to delineate the plasma membrane and with the cell-permeable DNA dye Draq5 to identify the nucleus. (F) Flow cytometric histograms for YoPro-1 and PI overlaying TDZD-8–treated (20 μM for 15 minutes) normal mononuclear cells or primary AML cells over untreated controls. Treated cells are represented with black line histograms and untreated controls with gray solid histograms. (G) Percent viability of primary AML cells pretreated with Z-VAD (□) for 1 hour before the treatment with TDZD-8 20 μM. Viability was determined 24 hours after the addition of TDZD-8. Error bars represent the SEM.