(Hyclone, Logan, UT, USA) and antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin; Sigma, St. Louis, MO, USA) at a density not exceeding 56105 cells/ml and maintained at 37uC in a humidified incubator with 5% CO2. To harvest BV-2 cells, cells were trypsinized (0.25% trypsin/EDTA in phosphate-buffered saline (PBS); Sigma, St. Louis, MO, USA), then centrifuged (400 g for 10 min) and resuspended in serum-free DMEM. Cells were counted with a hemocytometer and trypan blue staining (0.4% trypan blue in PBS; Sigma) showed more than 98% of the cells retained viability. Cells (approximately 16106 cells/ml) were seeded in six-well plates before being subjected to treatments. Resveratrol (Purity.99%; Kunming Pharmaceutical Corporation, Kunming, China) at 25, 50, and 100 mM was added 1 h before LPS (1 mg/ml) (from Escherichia coli, Sigma) stimulation. This time point was chosen to minimize the possibility of any direct interactions between resveratrol and LPS. Cell incubations ranged from 20 min–18 h, as indicated in the text. Cell cytotoxicity test The cells were seeded in a 24-well dish (16106 cells/ml) for 24 h before being exposed to resveratrol (25, 50, and 100 mM), resveratrol with LPS (1 mg/ml), resveratrol with rapamycin (10 nM), or resveratrol with LPS+rapamycin for 18 h. MTT solution (0.5 mg/ml) was then added to each well and the cells were incubated for 2 h at 37uC and in 5% CO2. Subsequently, the supernatant was removed and the formation of farmazan was solubilized with dimethyl sulfoxide (DMSO) and measured at 540 nm with a microplate reader. Assay of NO production Various treated BV-2 cells were plated in 48-well plates at a density of 16106 cells/ml for 8 h. NO production was monitored by measuring the nitrite content in culture medium as previously described [7]. The isolated supernatant was mixed with an equal volume of Griess reagent (0.1%N-1-naphthylethylenediamine dihydrochloride and 1% sulphanilamide in 5% phosphoric acid) and incubated at room temperature for 10 min. Absorbance was measured at 550 nm in a microplate reader. Sodium nitrite was used as a standard. Determination of PGE2 production Various treated BV-2 cells were treated for 8 h to permit cytokine production. The PGE2 concentration in the culture medium was quantified using a competitive enzyme immunoassay kit (R&D Systems Inc., MN, USA) in accordance with the manufacturer’s instructions. The production of PGE2 was measured relative to that observed after control treatment. Double-immunofluorescence labeling assay BV-2 cells derived from various treatments were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 15 min. After rinsing with PBS, the coverslips with adherent cells were used for double immunofluorescence labeling. BV-2 cells were incubated with DAPI (dilution 1:50,000; Sigma) plus goat anti- mouse iNOS (dilution 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-mouse COX-2 (dilution 1:500; Santa Cruz Biotechnology), goat anti-rabbit TNF-a polyclonal antibody (dilution 1:500; Chemicon, Temecula, CA,USA), goat anti-rabbit IL-1b (dilution 1:500; Chemicon), or goat anti-rabbit NF-kB/ RelA (dilution 1:500; Santa Cruz Biotechnology). Subsequently, the cells were incubated with TRITC-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. For negative controls, a set of culture slides was incubated under Resveratrol Inhibit LPS-Induced Activation by mTOR similar conditions without the primary antibodies. All images were captured with a fluorescence microscope (80i; Nikon, Tokyo, Japan). The results are representative of three independent experiments. Reverse transcription-polymerase chain reaction (RT-PCR) analysis After incubation, media was removed and BV-2 cells were washed with phosphate-buffered saline (PBS) twice. Total RNA was prepared from BV-2 cells by using the TrizolH reagent (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was prepared using reverse transcriptase originating from the SuperscriptTM-III kit (Invitrogen) with 2.5 mg total RNA and oligo dT. The sequences of PCR primers were as follows: iNOS, sense: 59- CTGCAGCA CTTGGATCAGGAACCTG -39, anti- sense: 59- GGGAGTAGCCTGTGTGCACCTGGAA -39; COX- 2, sense: 59-TTGAAGACCAGGAGTACAGC-39, antisense: 59-G GTACAGTTCCATGACA TCG-39; TNF-a, sense: 59-CGTCAG CCGATTTGCTATCT-39, antisense: 59-CGGACTCCGCAAA G TCTAAG-39; IL-1b, sense: 59-GCCCATCCTCTGTGACTCAT- 39, antisense: 59-AGGCCACAGG TATTTTGTCG-39; NF-kB/ RelA, sense: 59-GCGTACACATTCTGGGGAGT-39, antisense: 59-CCGAAGCAGGAGCTATCAAC-39; b-actin, sense: 59-AGC- CATGTACGTAGCCATCC-39, antisense: 59-GCTGTGGTGGT GAAGCTGTA-39. PCR amplification of the resulting cDNA template was conducted by using the following conditions for 45 (TNF-a, IL-1b, NF-kB/RelA and b-actin), 36 (COX-2) or 27 (iNOS) cycles. After an initial denaturation step at 95uC for 15 min, temperature cycling was initiated. Each cycle consisted of denatur- ation at 94uC for 15 sec, annealing at 60uC for 25 sec, and elongation at 72uC for 20 sec (TNF-a, IL-1b, NF-kB/RelA and b- actin). For COX-2, after an initial denaturation step at 95uC for 5 min, temperature cycling was initiated. Each cycle consisted of denaturation at 94uC for 30 sec, annealing at 57uC for 45 sec, and elongation at 72uC for 30 sec. For iNOS, after an initial denaturation step at 95uC for 5 min, temperature cycling was initiated. Each cycle consisted of denaturation at 94uC for 45 sec, annealing at 60uC for 45 sec, and elongation at 70uC for 1 min. PCR products were analyzed on 1% agarose gels and stained with 1 mg/ml ethidium bromide. Images were captured with a Gel Doc 2000 image analyzer (Bio-Rad, Richmond, CA, USA). The results are representative of three independent experiments. Western blot analysis BV-2 cells were incubated in a medium without 2% FBS for at least 4 h before treatments. They were harvested with ice-cold PBS and centrifuged at 16 0006 g for 5 min at 4uC. Stimulated cells were lysed in ice-cold lysis buffer [62.5 mM Tris–HCl, pH 6.8, 25% glycerol, 2% sodium dodecyl sulphate (SDS), 0.01% bromphenol blue and 5% b-mercaptoethanol]. Cell lysates were centrifuged at 16 0006g for 5 min at 4uC, then the supernatants were collected. Protein content was determined by using the BCA protein assay (Pierce, Rockford, IL, USA). Equal amounts of total cellular protein (50 mg) were loaded per lane onto 10% SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto immunoblot polyvinylidene difluoride membranes (Chemi- con). The membranes were blocked with 5% non-fat milk in Tris- buffered saline containing 0.1% Tween 20 (TBS-T) for 2 h and incubated separately with goat anti-rabbit antibodies for PTEN and phospho-PTEN, Akt and phospho-Akt, mTOR and phospho- mTOR, ERK1/2 and phospho-ERK1/2, JNK and phospho- JNK, p38 MAPK and phospho-p38 MAPK, phospho-IkB-a, phospho-CREB and b-actin antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA) that recognize PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e32195