Tetraarsenic Hexoxide Induces Beclin-1-Induced Autophagic Cell Death as well as Caspase-Dependent Apoptosis in U937 Human Leukemic Cells
Figure 6
Inhibition of As4O6-induced apoptosis and autophagy in U937 cells by N-acetylcysteine (NAC). NAC reduces the As4O6-induced autophagosome formation as well as As4O6-induced cell death. (a) U937 cells were treated with NAC (10 mM) 30 min before As4O6 (3 μM) for 24 h. The cells treated with As4O6 were stained with 5 μg/mL acridine orange for 17 min and collected in phenol red-free growth medium. Green (510–530 nm) and red (650 nm) fluorescence emission illuminated with blue (488 nm) excitation light was measured with a FACSCalibur (Becton Dickinson). (b) Sub-G1 DNA content was analyzed by flow cytometry. (c) To confirm apoptosis, the cells were stained with DAPI solution after fixation. Stained nuclei were then observed under fluorescent microscope using a blue filter (Magnification, X 400). (d) The cells were lysed and equal amount of the lysate was separated by SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were probed with the indicated antibodies and detected by an ECL detection system. To confirm equal loading, the blot was stripped of the bound antibody and reprobed with the anti β-Actin antibody. (e) The cell lysates from the cells treated with As4O6 were assayed for in vitro caspase-3activity using DEVD-pNA. The released fluorescent products were measured. The data are shown as means ± SD of three independent experiments. *P < 0.05 between the groups treated with and without As4O6, †P < 0.05 between the groups treated with and without NAC.
