Fig 1. ARC down-regulates antiapoptotic proteins and induces apoptosis in human leukemia cells.

A. ARC inhibits the growth of leukemia cells. Mid-log cells were treated with various concentrations of ARC for 48 hours and the surviving cells were counted and IC₅₀ value for each cell line was calculated. Leukemia cell lines CEM, HL-60, NB4 and U937 exhibited IC50s of 323 nM, 157 nM, 233 nM, and 187 nM, respectively B. ARC downregulates Mcl-1 expression, inhibits phosphorylation of Akt and induces caspase-3 cleavage in leukemia cells. The cells were treated as indicated, lysed and immunoblotted with specific antibodies as detailed. C. Caspase-8 inhibitor (Granzyme B inhibitor IV) does not protect U937 leukemia cells from ARC induced down regulation of Mcl-1 and apoptosis. The cells were treated with ARC and/or caspase-8 inhibitor as indicated for 24 hours, lysed and immunoblotted with specific antibodies. D. ARC induces mitochondrial injury in leukemia cells. The cells were stained with TMRE as detailed and the mitochondrial potential was measured by flow cytometry. E. Z-VAD-FMK, but not Z-VDVAD-FMK inhibits ARC-induced mitochondrial injury in U937 cells. The cells were treated with ARC with or without the inhibitors as indicated for 24 hours, stained with TMRE and analyzed by flow cytometry. F. Z-VAD-FMK, but not Z-VDVAD-FMK protects U937 cells from ARC-induced apoptosis. The cells were treated as indicated for 24 hours, stained with Annexin V-PE and 7-AAD and analyzed by flow cytometry as detailed.