CDDO-Me–induced decrease in the mitochondrial membrane potential and caspase activation in U937 cells.

Cells were assayed for apoptosis after 2, 4, and 6 hours of treatment with 1 μM CDDO-Me. (A) CMXRos assay (y-axis) to evaluate the reduction in the mitochondrial membrane potential. Cells expressing CMXRos below the threshold level were considered apoptotic. (C) Partial inhibition of CDDO-Me–induced loss of mitochondrial membrane potential by CyA. HL-60 cells were pretreated with the pharmacologic inhibitor of permeability transition CyA (10 μM) followed by CDDO-Me (1 μM) exposure for 2 or 4 hours. Loss of the mitochondrial membrane potential (ψ_m) was measured by CMXRos staining. No effect was observed from CyA alone (not shown). (D) HL-60 cells were exposed to 1 μM CDDO-Me for 2, 4, 6, and 8 hours. Caspase-3 cleavage was studied by Western blot analysis using antibody to caspase-3 (pro–caspase-3 at 32 kd and cleaved caspase-3 at 17 kd). (B) Activated caspase-3 (x-axis), as measured by conversion of Phi-Phi-Lux, and PI (y-axis). Cells showing a high conversion of Phi-Phi-Lux are considered apoptotic.