peroxidase-labeled anti-rabbit or anti-mouse IgG respectively (Super Signal detection kit, Pierce). RNA extraction, RT-PCR and real-time RT-PCR Total RNA was prepared using Trizol (Invitrogen) as described in the manufacturer’s protocol. For RT-PCR analysis, extracted RNA (1 mg) was digested with 2 U of DNase I (Invitrogen) for 30 min at 37uC. Samples were reverse transcribed to cDNA with 200 U of Moloney murine leukemia virus reverse transcriptase (RT) (Gibco-BRL Life Technologies), using 5 mg of random primers (Invitrogen) for 1 h at 45uC in a total volume of 20 ml. RT was inactivated at 95uC for 5 min. For each sample, an aliquot of DNase I-digested RNA, without RT, was used as a negative control for PCR amplification. The sequences of COX-2, COX-1, HSP70 and b-actin primers were as follows: COX-2, sense: 59- CAGCACTTCACGCATCAGTT-39, antisense: 59- CAGCAA- ACCGTAGATGCTCA-39; COX-1, sense: 59- AACATGGAC- CACCACATCCT-39, antisense: 59- TCCAGGGTAGAACTC- CAACG -39; HSP701A, sense: 59-CTACAAGGGGGAGAC- CAAGG-39, antisense: 59- TTCACCAGCCTGTTGTCAAA-39; b-actin, sense: 59-GCGCTCAGGAGGAGCAAT-39, antisense: 59-GCACTCTTCCAGCCTTCC-39. Amplification was per- formed using the following parameters: 94uC for 45 s, 60uC for 30 s, and 72uC for 45 s for 28 cycles for COX-2, COX-1 and HSP701A, and 24 cycles for b-actin. PCR products were electrophoresed alongside DNA Molecular Weight Marker IX (Roche Applied Science) in 2% agarose gels and then stained with ethidium bromide. PCR amplification was performed in a thermal cycler GeneAmp 2400 (Applied Biosystems), using Hot-Start Taq polymerase (QIAGEN). Real-time RT-PCR analysis was per- formed with ABI PRISM 7000 (Applied Biosystem, USA), using RealMasterMix ROX (Eppendorf) to prepare the reaction mixes. Primers used for real-time PCR of human genes were identical to the primers described above for semiquantitative RT-PCR. Relative quantities of selected mRNAs were normalized to b- actin in the same samples. Chromatin Immunoprecipitation (ChIP) assay Cells were fixed by adding formaldehyde (Sigma) to the medium to a final concentration of 1%. After 15 min, cells were washed with ice-cold phosphate-buffered saline (PBS) containing 1 mM phenylmethylsulfonyl fluoride, shaken for 20 min at 4uC in Lysis Buffer-1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, containing protease inhibitors) and harvested using a cell scraper. After centrifugation at 3000 rpm for 10 min the pellet was resuspended in Lysis Buffer-2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, containing protease inhibitors) and shaken at room temperature for 10 minutes. After centrifugation, nuclei were resuspended in Lysis Buffer-3 (50 mM Tris, pH 8.0, 1% SDS, 5 mM EDTA), and chromatin was sheared by sonication. After removal of nuclear debris by centrifugation at 13,000 rpm for 5 min at 8uC, lysates were diluted 10-fold with DB buffer (50 mM Tris pH 8.0, 5 mM EDTA, 200 mM NaCl, 0.5% NP40) and then precleared for 3 h using 80 ml of 50% salmon sperm-DNA saturated protein A (ss- proteinA)-agarose beads. Immunoprecipitation was carried out at 4uC overnight, and immune complexes were collected with ss- proteinA-agarose beads. Antibodies utilized included anti-HSF1 (Santa Cruz Biotechnologies) or pre-immune rabbit serum as control for non-specific interaction. After washing three times with high salt WB buffer (20 mM Tris, pH 8.0, 0.1% SDS, 1% NP-40, 2 mM EDTA, 0.5 M NaCl) and twice with low salt TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA), immunocomplexes were HSF1-Dependent COX-2 Induction by Heat eluted with TE containing 1% SDS. Protein-DNA cross-links were reverted by incubating at 65uC overnight. After proteinase K digestion, DNA was extracted with phenol-chloroform and precipitated with ethanol using 15 mg of tRNA as carrier. PCR was performed using the following primers: COX-2, sense: 59- AAATGAACAGTGCAACATGTGA-39, antisense: 59-CGTCT- GAAATTTGTTGGGAAA-39; HSP701A, sense: 59-CACTC- CCCCTTCCTCTCAG-39, antisense: 59-TTCCCTTCTGAGC- CAATCAC-39. For quantification of ChIP assays, samples from at least three independent experiments were analyzed by real time PCR. Data were normalized to the input DNA and DNA from untreated cells. PGE2 measurements PGE2 production in the supernatants of endothelial cells was determined by PGE2 EIA kit (Cayman Chemical Co.) according to the manufacturer’s instructions. The results are expressed as picograms per milliliter, calculated according to the PGE2 standard curve. All assays were performed in triplicate. Confocal microscopy HUVECs grown on coverslips were fixed with 4% paraformal- dehyde and permeabilized in 0,1% Triton X-100-PBS for 10 min. After blocking with 0,2% BSA, fixed cells were incubated with rabbit anti-COX-2 antibody (SC-1747 Santa Cruz Biotechnology) for 1 hour, and incubated with cy3-conjugated anti-rabbit IgG for 30 min at room temperature. Coverslips were mounted in Vectashield medium (Vector Laboratories). Images were acquired on a Leica confocal microscope TCS 4D system, equipped with a 100X 1.3–0.6 oil immersion objective. Statistical analysis Statistical analysis was performed using the Student’s t test for unpaired data. Data are expressed as the mean 6 S.D. of duplicate samples. P values of ,0,05 were considered significant. Supporting Information Figure S1 Kinetics of heat-induced binding of HUVEC HSF1 to an HSP70 HSE in vitro. HUVECs were subjected to heat shock at 43uC or left untreated (Control). After 20 and 40 min at 43uC (HS) or at the indicated times during recovery at 37uC (recovery), whole-cell extracts were analyzed for HSF DNA- binding activity by EMSA in a 4% polyacrylamide gel using an HSP70 HSE ideal probe [26]. Position of HSF-DNA binding complex (HSF), constitutive HSE binding activity (CHBA) and non-specific protein-DNA interactions (NS) are shown. (TIF) Figure S2 Analysis of in vitro and in vivo binding of HSF1 to the COX-2 promoter in heat-stressed human colon carcinoma cells. (A) HCT116 cells were subjected to heat shock at 43uC or left untreated (control). After 40 min at 43uC (HS) or at the indicated times during recovery at 37uC (recovery), nuclear extracts were analyzed for HSF DNA-binding activity by EMSA in a 3% polyacrylamide gel using the probe described in Fig. 5A. Position of HSF-DNA binding complex (HSF) and non-specific protein-DNA interactions (NS) are shown. (B) Specificity of HSF1-DNA binding complexes. Nuclear extracts from HCT116 cells subjected to heat shock at 43uC for 40 min were preincubated with different dilutions of anti-HSF1 polyclonal antibodies for 15 min before electromobility supershift assay. Position of HSF and NS are indicated as in A. (C,D) HCT116 cells were subjected to heat shock at 43uC or left untreated (C). PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e31304