Tetraarsenic Hexoxide Induces Beclin-1-Induced Autophagic Cell Death as well as Caspase-Dependent Apoptosis in U937 Human Leukemic Cells
Figure 5
Effects of Bcl-2 overexpression in U937 cells on the apoptosis and autophagy induced by As4O6. Overexpression of Bcl-2 suppresses the induction of Beclin-1 and LC3 conversion in response to As4O6 as well as As4O6-induced caspase-3 activation and PARP cleavages (a) U937/vector or U937/Bcl-2 cells were treated with 3 μM of As4O6 for 24 h. Sub-G1 DNA content was analyzed by flow cytometry. (b) 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). (c) 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). (d) The cells were lysed and equal amounts of proteins were then separated by SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with the indicated antibodies and detected by an ECL detection system. (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 U937/vector and U937/Bcl-2 cells.
