expression vector pCMV-LacZ (Invitrogen Life Technologies) expressing b-galactosidase. For each transfection, 2 mg of DNA was diluted into 50 ml of medium without serum. After the addition of 3 ml of LipofectAMINE into 50 ml Opti-MEM medium, the transfection mixture was incubated for 10 min at room temperature. Cells were washed with serum-free medium, the transfection mixture was added, and cultures were incubated for 24 hrs in the incubator. The next day, culture medium was replaced with fresh RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin mixture and resveratrol was added for desired times for the measurement of apoptosis and protein expression. Western Blot Analysis Western blots were performed as we described earlier [42]. In brief, cells were lysed in a buffer containing 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.5 mM EDTA, 1 mM EGTA, 1% SDS, 1 mM sodium orthovanadate, and a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mg/ml pepsta- tin A, 2 mg/ml aprotinin). Lysates were sonicated for 10 s, centrifuged for 20 min at 10,0006g and stored at 270uC. Equal amounts of lysate proteins were run on 10% SDS-PAGE and electrophoretically transferred to nitrocellulose. Nitrocellulose blots were blocked with 6% nonfat dry milk in TBS buffer (20 mM Tris-HCl (pH 7.4), and 500 mM NaCl), and incubated with primary antibody in TBS containing 1% bovine serum albumin overnight at 4uC. Immunoblots were washed three times (15, 5 and 5 min each) with TBST (TBS and 0.01% Tween 20). Immunoreactivity was detected by sequential incubation with horseradish peroxidase-conjugated secondary antibody and ECL reagents. Measurement of Apoptosis Cells were treated with resveratrol and apoptosis was measured by TUNEL assay as per manufacturer instructions (EMD Biosciences). Caspase-3 Assay Cells (36104 per well) were seeded in a 96-well plate with 200 ml culture medium. Approximately 16 h later, cells were treated with various doses of resveratrol to induce apoptosis. Casapse-3 activity was measured by a fluorometer as per manufacturer’s instructions (EMD Biosciences). Cell Cycle Analysis PANC-1 cells were transfected with either scrambled or FKHR/FKHRL1/AFX-shRNA (FOXO-shRNA) plasmids. FKHR/FKHRL1/AFX-shRNA (FOXO-shRNA) plasmids have been described elsewhere [28]. Transfected cells were treated with resveratrol (10 mM) for 24 h. Cell cycle analysis was performed as we described elsewhere [28]. Clonogenic Assay Pancreatic cancer cells (104 cells) were mixed with 0.3% agarose and layered over a solid base of 0.5% agarose in RPMI in the absence or presence of resveratrol in 33-mm dishes. The cultures were incubated in humidified 5% CO2, 95% air at 37uC for 21 days and then stained with the nitro blue tetrazolium. Colonies of .120 mm in diameter were counted using a microscope. AKT Enzyme Assays Cells were lysed in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% (w/v) Triton X-100, 10% glycerol, FOXO-Dependent Regulation of Tumorigenesis 1 mM EDTA, 1 mM dithiothreitol, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml bacitracin, 1 mM Na3VO4, 30 mM NaPPi, 10 mM NaF, 100 nM okadaic acid). Following centrifugation, the supernatants were incubated for 2 h at 4 uC with protein A-Sepharose beads coated with 2.5 ml of anti- Akt antibody. Immunoprecipitates were washed three times with the lysis buffer and twice with the kinase assay buffer (50 mM Tris- HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol) and assayed using GSK-3 peptide (GRPRTSSFAEG) as substrate as described [43]. Evaluation of mRNA expression levels by quantitative Real Time-PCR For the quantification of gene amplification, Real-time PCR was performed using an ABI 7300 Sequence Detection System in the presence of SYBR- Green. Briefly, RNA isolated with or without treatment with the chromatin modifiers using TRIzol (Life Technologies) were reverse transcribed. cDNA reactions were amplified with QPCR SYBR Green Mix (Applied Biosystems) and the following gene-specific primers: Sirt1 (59-tgt ggt aga gct tgc att ga-39, 59- gcc tgt tgc tct cct cat ta- 39) Sirt2 (59-aag act gtc aga gcc tgg tg-39, 59- ata atc atc ccc agg aaa gg-39) Sirt3 (59-tgg aat tgg tga cct agg aa-39, 59-acc tgt aat ccc agt tac cc-39) HK-GAPD (59-gag tca acg gat ttg gtc gt-39, 59-ttg att ttg gag gga tct cg-39) Target sequences were amplified using incubated at 95uC for 10 min, followed by 40 cycles of 95uC for 15 s and 60uC for 1 min. HK-GAPD is used as endogenous normalization control. All assays were performed in triplicate and were calculated on the basis of DDCt method. Luciferase Assay Pancreatic cancer cell lines were transfected with empty vector, FOXO1-TM, FOXO3a-TM or FOXO4-TM along with reporter plasmids, p6xDBE-luc and pRL-TK [44]. The FOXO expression vectors (wild type and phosphorylation deficient mutants) and FOXO-luciferase constructs have been described elsewhere [44]. After 24 h, transfection medium was replaced with culture medium and cells were treated with resveratrol (0–20 mM). After incubation of 24 h, the relative luciferase activity, i.e. firefly enzyme activity divided by that of the Renilla enzyme, was determined using Dual Luciferase Reporter Assay System (Promega, Madison, WI) according to the manufacturer’s protocol. Electrophorectic mobility shift assay (EMSA) FOXO probes were end-labeled with [c-32P] dATP by incubating oligodeoxyribonucleotide strands with 56 reaction buffer and 10 U T4 polynucleotide kinase for 1 h at 37uC. Then labeled oligonucleotides were allowed to anneal at room temperature for 10 min and 20 mg protein from each sample was used in 25 ml binding reactions, which consisted of 1 mg poly dI-dC, in 56 binding buffer (50 mM Tris HCl; pH 8.0, 750 mM KCl, 2.5 mM EDTA, 0.5% Triton-X 100, 62.5% glycerol (v/v) and 1 mM DTT). To determine specificity of DNA binding, samples were incubated with or without 20 ng of unlabeled competitor DNA for 10 min at room temperature. Then 0.1 ng of labeled probe was added and samples were further incubated for 20 min at room temperature. Samples were separated on a 5% non-denaturing polyacrylamide gel in 0.5% TBE and visualized by autoradiography. PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e25166