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A. E2 (PGE2) production of peritoneal macrophages (pMs). pMs with SIRT1 overexpression displayed improved phagocytosis and tumoricidal functions, which are associated with stressed out PGE2. Furthermore, SIRT1 protein level was up-regulated in CR mouse pMs, whereas elevated SIRT1 decreased COX-2 expression and improved PGE2-related macrophage functions that were reversed following inhibition of SIRT1 deacetylase activity. Thus, our results indicate that SIRT1 may be a mediator of CR-induced macrophage regulation, and its deacetylase activity contributes to the inhibition of AP-1 transcriptional activity and COX-2 expression leading to amelioration of macrophage function. and access to water and feed for any 1-week acclimation period and randomly assigned to two groups. A group of mice was given (AL) access to water and feed as control group, and another group of mice was given 60% of daily calorie intake of control mice as CR group. Composition of the diet and the feeding protocol was explained elsewhere in detail (24). All of the animal experiments were performed in accordance with institutional guidelines. Thioglycollate-elicited pMs were obtained from mice that had been injected with 2 ml of 3% (w/v) thioglycollate 5 days before sacrifice. The isolated cells were incubated in RPMI 1640 medium made up of 10% fetal bovine serum for 3 h followed by washing three times with phosphate-buffered saline to remove nonadherent cells before treatment. Reagents and Antibodies The reagents, including PMA, thioglycollate, resveratrol (RSV), and Sirtinol were purchased from Sigma-Aldrich. Anti-HA and anti–actin monoclonal antibodies were from Sigma-Aldrich, and anti-Fos, -Jun, and -hSIRT1 (human SIRT1) rabbit polyclonal antibodies were from Santa Cruz Biotechnology. Anti-mSIRT1 (mouse SIRT1) and anti-acetyl-lysine antibody were from Upstate, and anti-COX-2 antibody was from Cayman Chemicals. Plasmid and Computer virus Production The AP-1-luc plasmid was obtained by the insertion of a double-stranded oligonucleotide representing seven AP-1 binding sites into the pTK-luc plasmid. The full-length and deletion mutant c-Fos and c-Jun expression vectors were constructed by inserting the human c-Fos or c-Jun cDNA in the frame of pcDNA3.1 vector. SIRT1 expression vectors were gifts PROTAC ERRα ligand 2 from Dr. Ishikawa (25). The mouse COX-2 promoter plasmid made up of a 1068-bp fragment, ?1003 to +65 relative to the transcription start site, was subcloned into pGL3 vector (COX-2-luc). The wild type COX-2 promoter was altered using a site-directed mutagenesis kit (Promega). The AP-1 binding element (5-ACA GAG PROTAC ERRα ligand 2 TCA C-3 from ?95 to ?86) was modified to 5-ACA TTA TAA C-3. The underlined sequences denote the mutated site, and it was confirmed by DNA sequencing. GST fusion proteins were expressed in strain BL-21 by using the pET42a vector system. Replication-defective adenoviral vectors expressing SIRT1 (Ad-SIRT1) or control green fluorescent protein (Ad-GFP) were prepared using the AdEasy vector kit (Quantum Biotechnologies) as PROTAC ERRα ligand 2 explained in the supplier’s protocol. The adenovirus-mediated knockdown of SIRT1 (Ad-SIRT1 RNAi) and control vector (Ad-U6) were generated using the same system. The RNAi sequence was reported previously (26). pMs were infected with the above adenovirus for 36 h and were cultured in new RPMI 1640 for further treatment. Transfections and Luciferase Assays HEK293 cells and RAW264.7 were grown as recommended by ATCC and were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Luciferase assays were performed using a dual luciferase reporter assay system (Promega). Luciferase activity was normalized by transfection efficiency using pRL-TK reporter (Promega) as an internal control (30 ng). The results are expressed as percentages of relative luciferase activity of the control group, which was set as 100%. Co-immunoprecipitation, AP-1 Acetylation Assays, GST Pulldown Assays, and Western Blotting The cells were collected, and the proteins were solubilized in IP buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% protease inhibitor combination) at 4 C. One milligram of lysated protein was incubated with specific antibodies and precipitated with protein A- or G-agarose (Upstate Biotechnology). Purification of GST fusions (GST-Fos-HA, GST-Jun-HA, and control GST-HA) and maltose-binding protein-tagged SIRT1 from bacterial lysates was performed, and the binding assays H3FH were carried out using Glutathione-Sepharose (GE Healthcare) according to the manufacture’s instructions. Precipitated proteins or total lysates were separated in 12% PAGE followed by immunoblotting with specific antibodies. The blots were then incubated with horseradish peroxidase-conjugated secondary antibody and exposed to ECL reagent for detection of protein expression. DNA-Protein Binding Assays PROTAC ERRα ligand 2 The probe is usually a biotinylated, double-stranded oligonucleotide corresponding to human promoter sequence ?72/?18, which contains the binding site of AP-1 (5-ACA GAG TCA C-3). Four micrograms of probe were incubated with 400 g of nuclear protein extract and 30 l of streptavidin-agarose bead slurry at room heat for 2.