CPS45 induces rapid caspase-independent cell death reversed by antioxidants. (A) FACS measurement of 10 μM CPS45–induced cell death by annexin and PI staining after 0 to 3 hours of drug exposure. Percentages indicate percentage of cells in the respective quadrants. (B) FACS analysis of the reversal of cell killing by the broad-spectrum caspase inhibitor z-VAD (200 μM), 5 μM ebselen, 20 mM NAC, 10 μM DPI, or 50 μM MnTBAP. Changes are shown as a percentage of maximum PI-positive cells in the presence of 10 μM CPS45 alone. (C) Kinetic profile of ROS generation, intracellular calcium elevation, and loss of mitochondrial membrane potential at 15-minute intervals during 1 hour of treatment with 10 μM CPS45. Data represents means of triplicate determinations normalized to the percentage of maximum. (D) Immunoblot analysis of caspase 7 (nuclear and cytosolic), caspase 3 (nuclear), and PARP (nuclear) cleavage in PHA/PMA-stimulated Jurkat cells untreated or treated for 8 hours with 10 μM CPS45. Arrows indicate activated caspase cleavage products.