Figure 2.

PGE₂ increases HIF-1 activity by suppressing SIRT1 levels in human breast adipose stromal cells. A–E, human breast ASC cell line was used. A and B, the bottom panels represent primary human breast ASCs. A and B, cells were treated with the indicated concentrations of PGE₂ for 24 h. Cells were then harvested, and lysates were subjected to Western blotting. C and D, cells were transfected with 2 μg of control vector or SIRT1 expression vector as indicated. C (top), cells were harvested, and Western blotting was performed for SIRT1 and β-actin to confirm overexpression. C (bottom) and D, cells were treated with vehicle or 500 nm PGE₂ for 24 h. Cell lysates were prepared and subjected to Western blotting, and the blots were probed as indicated. E, cells were transfected as indicated with 0.9 μg of HRE-luciferase and 0.2 μg of psvβ-gal constructs. Cells labeled Control Vector also received 0.9 μg of expression vector; cells labeled SIRT1 Vector also received 0.9 μg of SIRT1 expression vector. 24 h after transfection, cells were treated with vehicle (control) or 500 nm PGE₂ for 24 h. Cells were harvested, and luciferase activity was measured. Luciferase activity was normalized to β-gal activity. Means ± S.D. (error bars) are shown, n = 6. *, p < 0.001 versus PGE₂-treated cells expressing control vector.