Figure 1.

2ME markedly induces apoptosis and mitochondrial injury in U937human leukemia cells in a dose- and time-dependent manner. (a) U937cells were treated without or with various concentrations of 2ME as indicated for 24 h. (b) U937cell were treated with 4 μM 2ME for 3, 6, 12, and 24 h. Cells were stained with annexin V/propidium iodide (PI), and apoptosis was determined using flow cytometry as described in Materials and methods. In separate experiment, cells were stained with DiOC₆, and reduction in Ψ_m was determined by monitoring uptake of DiOC₆ using flow cytometry as described in Materials and methods.‘Low’Ψ_m values are expressed as the percentage of cells exhibiting a diminished mitochondrial membrane potential. The values obtained from annexin V/PI and DiOC₆ assays represent the mean±s.d. for three separate experiments. (c) U937cells were treated without or with various concentrations of 2ME as indicated for 24 h. (d) U937cell were treated without or with 4 μM 2ME for 3, 6, 12, and 24 h. After treatment of U937cells with the indicated 2ME concentration or the indicated interval, total cellular extracts, cytosolic S-100 fractions (cytochrome c, AIF), and nuclear extracts (AIF) were prepared and subjected to Western blot assay using antibodies against PARP, cleaved-caspase-3, caspase-8, caspase-9, AIF, and cytochrome c. AIF(N) ¹/4 AIF in the nuclear fraction. Each lane was loaded with 30 μg protein. Blots were subsequently stripped and reprobed with antibody against β-actin to ensure equivalent loading and transfer. Two additional studies yielded equivalent results