Journal: The Journal of Biological Chemistry

Article Title: Prostaglandin E 2 down-regulates sirtuin 1 (SIRT1), leading to elevated levels of aromatase, providing insights into the obesity–breast cancer connection

doi: 10.1074/jbc.RA118.005866

Figure Lengend Snippet: 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.

Article Snippet: Primary human breast ASCs and media were purchased from Zen-Bio.

Techniques: Activity Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Over Expression, Luciferase, Construct, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Prostaglandin E 2 down-regulates sirtuin 1 (SIRT1), leading to elevated levels of aromatase, providing insights into the obesity–breast cancer connection

doi: 10.1074/jbc.RA118.005866

Figure Lengend Snippet: Overexpression of SIRT1 inhibits PGE2-mediated induction of aromatase. Human breast ASC cell line was used except in D, where primary human breast ASCs were employed. A and B, cells were treated with 500 nm PGE2 for 0–24 h as indicated. C–E, cells were either untransfected (Control) or transfected as indicated. Cells were then treated with vehicle or 500 nm PGE2 for 24 h. A–D, cell lysates were subjected to Western blotting, and the blots were probed as indicated. E, aromatase mRNA levels were determined by quantitative PCR. F, cells were transfected with 0.9 μg of aromatase promoter PII-luciferase and 0.2 μg of psvβ-gal constructs. In addition, as indicated, cells received 0.9 μg of control expression vector or SIRT1 expression vector. 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. G, cells were either untransfected or transfected with 0.9 μg of control vector or SIRT1 expression vector. Subsequently, cells were treated with vehicle or 500 nm PGE2 for 3 h. ChIP assays were performed. Chromatin fragments were immunoprecipitated with antibodies against HIF-1α, and the aromatase promoter was amplified by real-time PCR. DNA sequencing was carried out, and the PCR products were confirmed to be the aromatase promoter. This promoter was not detected when normal IgG was used or when antibody was omitted from the immunoprecipitation step. In E–G, means ± S.D. (error bars) are shown (n = 6). *, p < 0.001.

Article Snippet: Primary human breast ASCs and media were purchased from Zen-Bio.

Techniques: Over Expression, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Luciferase, Construct, Expressing, Plasmid Preparation, Activity Assay, Immunoprecipitation, Amplification, DNA Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Prostaglandin E 2 down-regulates sirtuin 1 (SIRT1), leading to elevated levels of aromatase, providing insights into the obesity–breast cancer connection

doi: 10.1074/jbc.RA118.005866

Figure Lengend Snippet: PGE2 down-regulates SIRT1 expression in human breast ASCs. A, cells were treated with the indicated concentrations of PGE2 for 24 h. Total RNA was isolated, and quantitative PCR for SIRT1 was performed. B, cells were transfected with 1.8 μg of full-length SIRT1 promoter and 0.2 μg of psv-β-gal constructs. 24 h after transfection, cells were treated with vehicle or 500 nm PGE2 for 24 h. Cells were harvested, and luciferase activity was measured. Luciferase activity was normalized to β-gal activity. C and D, cells were treated with vehicle (control) or the indicated concentrations of butaprost or CAY10684 for 24 h. E and F, cells were pretreated with vehicle, PF04418948, or ONO AE3 208 for 2 h. Subsequently, the cells were treated with vehicle, 500 nm PGE2, or 500 nm PGE2 plus the indicated concentrations of PF04418948 (E) or ONO AE3 208 (F) for 24 h. C–F, cell lysates were prepared and subjected to Western blotting. A and B, means ± S.D. (error bars) (n = 6). *, p < 0.001.

Article Snippet: Primary human breast ASCs and media were purchased from Zen-Bio.

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Transfection, Construct, Luciferase, Activity Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Prostaglandin E 2 down-regulates sirtuin 1 (SIRT1), leading to elevated levels of aromatase, providing insights into the obesity–breast cancer connection

doi: 10.1074/jbc.RA118.005866

Figure Lengend Snippet: ATF3 is important for PGE2-mediated down-regulation of SIRT1 in human breast ASCs. A–G, a human breast ASC cell line was used. A, cells were pretreated with vehicle or the indicated concentrations of H89, a PKA inhibitor, for 2 h and then treated with vehicle, 500 nm PGE2, or 500 nm PGE2 plus the indicated concentrations of H89 for 24 h. B, cells were treated with the indicated concentrations of PGE2 for 2 h. Cell lysates were subjected to Western blotting, and the blots were probed as indicated. C, cells were pretreated with vehicle or the indicated concentrations of H89 for 2 h and then treated with vehicle, 500 nm PGE2, or 500 nm PGE2 plus the indicated concentrations of H89 for 24 h. D, cells were transfected with 0.9 μg of full-length SIRT1 promoter and 0.2 μg of psv-β-gal constructs. In the column labeled Control siRNA, cells received 0.9 μg of control siRNA, and in the column labeled ATF3 siRNA, cells received 0.9 μg of ATF3 siRNA. 24 h later, cells were treated with vehicle (control) or PGE2 for 24 h. Cells were harvested, and luciferase activity was measured. Luciferase activity was normalized to β-gal activity. Inset, Western blotting was performed after transfecting ASCs with control or ATF3 siRNA. E, the top panel represents different deletions of SIRT1 promoter used. Full-length promoter and deletion 1 (Del1) contain a CRE site, whereas deletion 2 (Del2) lacks a CRE site. Cells were transfected with 1.8 μg of each of three SIRT1 promoter-luciferase constructs and 0.2 μg of psv-β-gal for 24 h. Subsequently, cells were treated with vehicle (control) or 500 nm PGE2 for 24 h. Cells were then harvested, and luciferase activity was measured. Luciferase activity was normalized to β-gal activity. F, ChIP assay was performed. Cells were treated with vehicle (control) or 500 nm PGE2 for 3 h. Chromatin fragments were immunoprecipitated with antibodies against ATF3, and the SIRT1 promoter was amplified by real-time PCR. DNA sequencing was carried out, and the PCR products were confirmed to be the SIRT1 promoter. This promoter was not detected when normal IgG was used or when antibody was omitted from the immunoprecipitation step. G, cells were either untransfected or transfected as indicated with 2 μg of siRNA to GFP (control siRNA) or ATF3 siRNA. 48 h after transfection, cells were treated with vehicle (control) or 500 nm PGE2 for 24 h. Cell lysates were subjected to Western blotting, and the blots were probed as indicated. D–F, means ± S.D. (error bars); n = 6. *, p < 0.001.

Article Snippet: Primary human breast ASCs and media were purchased from Zen-Bio.

Techniques: Western Blot, Transfection, Construct, Labeling, Luciferase, Activity Assay, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, DNA Sequencing