ROS develop within minutes in an intracellular compartment in CPS45-treated leukemic cells and selectively cause damage to the mitochondria of leukemic cells. (A) Jurkat T cells were bound to poly-lysine–coated slides and preincubated with the oxidation-sensitive fluorescent dye DCFDA; fluorescence was captured by live-cell imaging on a Zeiss 510 confocal microscope (Carl Zeiss, Oberkochen, Germany) using a 100×/1.3 NA oil objective at 10-second intervals from 0 to 36 minutes after the addition of 10 μM CPS45. Captured images were annotated using Adobe Photoshop (Adobe, San Jose, CA). Arrows indicate intracellular origin of ROS in the region of the ER and associated mitochondria (Video S1). (B) Electron microscopic (EM) images of Jurkat cells (top left) and PBMCs (bottom left and bottom right) treated with 10 μM CPS45 for 4 hours. Higher magnification of Jurkat cells treated for 1 hour with CPS 10 μM 45 (top right) contrasted against PBMCs treated for 4 hours with 10 μM CPS45 (bottom right). Scale bar equals 0.5 or 2.5 μm as indicated. Arrowheads indicate mitochondria. (C) Schematic representation of the proposed mechanism of CPS45-induced cell death in leukemic cells. Entry of CPS45 initiates a cascade of molecular events beginning with elevation of ROS that originates in the ER and mitochondrial (Mito) compartment. The elevated ER stress leads to elevated calcium and subsequent NFAT induction. The ROS-induced action at the mitochondria is self-amplifying. These events culminate in mitochondrial dysfunction, elevation of stress-response genes, and cell death.