Detection of apoptosis by flow cytometry: histogram analysis. A549 cells were transfected with HIF-1α siRNA vector or HIF-1 expression plasmid. Cells were allowed to recover in regular culture medium for 20 h after transfection, then exposed to normoxic or hypoxic environment for 24 h with or without additional HEPES (25 mM) or the glycolytic inhibitor, 2-DG (5 mM) (n = 5 in each group). Cells were collected and stained with Apo2.7 monoclonal antibody (7A6). Shown are representative results from one of five independent experiments. siRNA: A549 cells transfected HIF-1α siRNA vector, Mock: mock-transfected, HIF: A549 cells transfected with HIF-1α expression plasmid, HIF+2-DG: Cells transfected with HIF-1α expression plasmid and cultured with 2-DG, HIF+HEPES: Cells transfected with HIF-1α expression plasmid and cultured with medium containing HEPES.