ROS up-regulate glycolysis in hepatoma cells under hypoxia. (a) Hypoxia stimulates ROS generation. SMMC-7721 hepatoma cells were exposed to 0.5% O2, 2% O2, and 5%O2 for 24 h, and ROS generation was determined by DCF fluorescence using flow cytometry. (b) LDH activity increases in response to hypoxia. SMMC-7721 hepatoma cells were incubated for 24 h under indicated hypoxic conditions, and LDH activity was measured as described in "Materials and methods". (c) The effect of antioxidant α-LA on LDH activity under hypoxia. Cells were pre-treated with or without 5 mmol/L α-LA and exposure to 2% O2 for 24 h. Scavenging ROS by α-LA attenuates hypoxia-induced LDH activity. (d) ROS is involved in hypoxia-induced HIF-1α stabilization. Cells were subjected to different hypoxic condition for 24 h, in some cases cells were pre-treated with 5 mM α-LA. Nuclear HIF-1α levels were assessed by Western blot as described in Materials and methods. β-actin shows the internal reference for semiquantitative loading in each lane. The present blots are representative of three experiments. Columns, data were normalized to 21% O2 normoxia controls (a, b, c) and represent the means ± S.D. of four independent experiments. *, P < 0.05 versus normoxia. **, P < 0.05 versus cells in the absence of α-LA treatment under 2% O2 hypoxia.