EEAA down-regulates HIF signaling via enhanced proteasomal degradation. (A) Effect of EEAA on HIF signaling. NIH3T3/HIF-luc cells were pre-treated with vehicle (DMSO) or with increasing concentrations of EEAA (1–100 μ g/mL) for 2 h before exposure to hypoxia (1% O2). After 24 h, intracellular luciferase activity was quantified. Relative luciferase activity was determined based on comparing with mock under normoxia (20% O2). The data are presented as the relative means ± S.D. of at least three independent experiments compared with vehicle. ***P < 0.001 compared with the vehicle groups. (B) Effect of EEAA on HIF protein expression under gas- or chemical-induced hypoxia. NCI-H460 cells were pre-treated with 100 μ g/mL EEAA for 2 h before exposure to hypoxic gas (1% O2) or to 150 μ M CoCl2. After 24 h of incubation, WCL was prepared and subjected to western blot analysis. (C) Enhanced proteasomal degradation of HIF-1α via EEAA. NCI-H460 cells were pre-treated with a combination of vehicle (DMSO) or 10 μ M MG132 and 100 μ g/mL EEAA for 2 h before exposure to hypoxic gas (1% O2). After 24 h of incubation, intracellular HIF-1α levels were determined through western blot analysis. β-actin was used as a loading control.