Skip to main content
. 2012 Aug 29;287(42):35161–35169. doi: 10.1074/jbc.M112.367367

FIGURE 3.

FIGURE 3.

Clofibrate suppresses hypoxia-induced HIF-1α expression and activity through PPARα activation. A, MCF-7 cells were transfected with the pGL3-HRE-luciferase reporter construct. Cells were treated with 500 μm clofibrate, 100 μm fenofibrate, and 20 μm troglitazone for 4 h prior to placement in a hypoxia chamber for 16 h. Cell lysates were prepared, and luciferase activity was assayed. Data (mean ± S.E., n = 3) are expressed as percentages of the luciferase activity detected in untreated cells under normoxia. **, p < 0.01 compared with untreated cells using one-way ANOVA, followed by Dunnett's analysis. B, MCF-7 cells were treated with 500 μm clofibrate, 100 μm fenofibrate, and 20 μm troglitazone for 4 h prior to placement in a hypoxia chamber for 16 h. Cell lysates were prepared, and Western blotting was performed using antibodies against HIF-1α and β-actin. Shown are representative images of three individual experiments. C, MCF-7 cells were transfected with the pGL3-HRE-luciferase reporter construct. Cells were pretreated with 5 μm GW6471 or 10 μm GW9662 for 15 min prior to addition of 500 μm clofibrate for another 4 h. The cells were then placed into a hypoxia chamber for 16 h. Cell lysate was prepared, and luciferase activity was assayed. Data (mean ± S.E., n = 3–5) are expressed as percentages of the luciferase activity detected in untreated cells under normoxia. *, p < 0.05 compared with untreated cells using one-way ANOVA, followed by Dunnett's analysis. D, MCF-7 cells were pretreated with 5 μm GW6471 or 10 μm GW9662 for 15 min prior to addition of 500 μm clofibrate for another 4 h. The cells were then placed into a hypoxia chamber for 16 h. Cell lysates were prepared, and Western blotting was performed using antibodies against HIF-1α and β-actin. Shown are representative images of three individual experiments. E, MCF-7 cells were transfected with individual PPARα siRNAs (100 pmol/ml; siRNA-a or siRNA-b) for 48 h. Cell lysates were prepared, and Western blotting was performed using antibodies against PPARα and β-actin. F, MCF-7 cells were transfected with a pool of PPARα siRNAs (100 pmol/ml; siRNA-a, siRNA-b, and siRNA-c) prior to treatment with 500 μm clofibrate for 4 h. The cells were then placed into a hypoxia chamber for 16 h. Cell lysates were prepared, and Western blotting was performed using antibodies against HIF-1α, PPARα, and β-actin. Shown are representative images of three individual experiments.