Materials and Methods Cell culture and treatments Primary human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex Bio Science (Walkersville, MD, USA) and grown in EGM-2 complete medium (Cambrex Bio Science) according to the manufacturer’s instructions. Cells were incubated at 37uC with 5% CO2 in non-coated 100 mm BD FalconTM cell culture dishes (BD Biosciences Discovery Labware). Culture medium was changed at day 1 and every 2 days thereafter. Monolayer cell confluence was achieved after 4–6 days of culture. All experiments were performed using HUVEC passage 2–5. Human colorectal adenocarcinoma (HCT116) and lymphoblas- toid (Jurkat) cells (American Type Culture Collection) were maintained in McCoy’s 5 A (HCT116) or RPMI 1640 (Jurkat) medium supplemented with 10% FCS, 2 mM glutamine, and antibiotics. Human peripheral blood monocytes, isolated and purified from buffy coat of healthy blood donors (kindly provided by Prof. Adorno, Haematology Division, University of Rome Tor Vergata) as described elsewhere [10], were grown for 24 h in RPMI 1640 medium, supplemented with 10% FCS and antibiotics as indicated above. For heating procedures, cells were subjected to heat shock at the indicated temperatures in a precision water bath-W14 (Grant Instruments). For long exposure at febrile temperatures HUVECs were incubated in automatic CO2 incubators certified to have ,0.2uC temperature variation and calibrated for each experiment using an electronic thermom- eter. Selective COX-2 inhibitor NS-398, tetradecanoylphorbol 13- acetate (TPA) and interleukin 1b (IL-1b) (Sigma) were dissolved in dimethylsulfoxide (DMSO) and diluted in the culture medium immediately before use. Control cells received the same amount of vehicle. Cloning of COX-2 promoter, preparation of 59 deletion constructs and mutants To generate the COX-2-PGL3 vector indicated as full-length (FL) in this study, a pair of gene-specific primers (sense: 59- CGGGCTAGCTGTGCTTTCGTTTATAGCCTCA-39; anti- sense: 59-GCTAAAGCTTGGGTAGGCTTTGCTGTCTGA- 39) was designed to amplify the COX-2 gene promoter region (spanning from 23120 upstream of the gene transcription start site to +110) from human genomic DNA (Promega) by using Phusion High-Fidelity DNA polymerase (Finnzymes). The reaction product was analyzed by agarose gel electrophoresis, digested with NheI and HindIII, and inserted upstream of the luciferase gene of the pGL3Basic vector (Promega) to generate the FL-COX2-PGL3 (FL) construct. The -2221-COX2-PGL3 (22221, Del1), -1787- COX2-PGL3 (21787, Del2) and -1396-COX2-PGL3 (21396, Del3) serial deletion mutants were generated as described above. Oligos used were: for 22221, Del1 construct, sense: 59- CGGGCTAGCACCAGCATCCCAAATGTACC-39; for -1787, Del2 construct, sense: 59- CGGGCTAGCAACATGGCTTCTA- ACCCAAA-39; for 21396, Del3 construct, sense: 59- CGGG- CTAGCGCTGTCATTTTCCTGTAATGC-39. The antisense oligo used was the same as that used for the construction of the FL construct. The D-HSE-COX2-PGL3 (D-HSE), D-GATA- COX2-PGL3 (D-GATA) and D-HSE-D-GATA-COX2-PGL3 (D- HSE-D-GATA) mutant constructs were generated by using the QuikChange Site-Directed Mutagenesis Kit following the manu- facturer’s instructions (Stratagene). Oligos used were: for D-HSE construct, sense: 59-GCAGAAGGGGGCAGTAAAACTAGC- TCTGGAAG-39, antisense: 59-CTTCCAGAGCTAGTTTTAC- TGCCCCCTTCTGC-39; for D-GATA construct, sense: 59-AC- ACAAAATCTGTCTTCCTAGCATCTTAGGAAAAAAGTT- HSF1-Dependent COX-2 Induction by Heat G-39, antisense: 59-CAACTTTTTTCCTAAGATGCTAGGAA- GACAGATTTTGTGT-39. For the D-HSE-D-GATA construct, the same oligos used for D-GATA were used on the D-HSE construct. The nucleotide sequence of each construct was verified by DNA sequencing. Recombinant retroviral vectors For HSF1 knockdown experiments, we used the RNAi-pSuper- retro vector, containing a puromycin resistance gene for selection of stable transfectants. The sequence of human HSF1 gene was selected as reported before [55]. Retroviruses were produced by transfection of 293T cells with plasmids expressing retroviral proteins Gag-Pol, G (VSV-G pseudotype), pSUPER-retro and HSF1i-pSUPER-retro constructs using Lipofectamine 2000 (In- vitrogen). At 48 h after transfection, supernatants containing the retroviral particles were collected and frozen at 270uC until use. HCT116 cells were infected with diluted supernatant in the presence of 8 mg/ml Polybrene overnight, and then selected with puromycin (1 mg/ml) 48 h after infection. After 10 days in selective medium, two pools referred to as pHCT116-pSUPER- retro (pHCT116-retro) and pHCT116-pSUPER-retro-HSF1i (pHCT116-HSFi) were isolated. The puromycin selective pressure was removed 24 hours before experimental procedures. Cell transfection and reporter assays Transfections were performed using FuGENE HD Transfection Reagent (Roche) for HUVEC cells and LipofectAMINE Plus reagent (Invitrogen) for HCT116 cells according to the manufac- turer’s protocols. For reporter gene experiments, the different COX-2 constructs were co-transfected with a control plasmid (pRL-TK encoding Renilla luciferase; Promega) to normalize transfection efficiency. Transfected cells were grown for 16 h before heat treatment and luciferase activity of quadruplicate samples was measured in a Microplate Luminometer (Wallac- Perkin Elmer) using Dual-Luciferase kit (Promega). COX-2 promoter firefly luciferase activity was normalized to Renilla luciferase activity in the same sample. Electrophoretic Mobility Shift Assay (EMSA) The 100 bp DNA probe (shown in Fig. 5A) was prepared by PCR amplification of the COX-2 promoter region containing the HSF1 binding site (from 22579 to 22480) using the following primers: sense: 59- AAAATCTGTCTGGCAATCTTCC-39, an- tisense: 59- TCTTCCAGAGCTTTCTGTTGAG-39. This PCR product was labeled at both ends using [c32-P]ATP plus T4 nucleotide kinase. Whole-cell or nuclear extracts were prepared as described [56]. Whole-cell (15 mg) and nuclear (5 mg) extracts were incubated with a 32P-labeled probe followed by analysis of DNA- binding activity by EMSA. Binding reactions were performed as described [57]. Complexes were analyzed by nondenaturing 3% polyacrylamide gel electrophoresis. Quantitative evaluation of HSF-HSE complex formation was determined by Typhoon 8600 imager (Molecular Dynamics, Amersham Biosciences) with the use of ImageQuant (Amersham Biosciences). Western blot analysis Equal amounts of protein (35 mg/sample) from whole-cell extracts were separated by SDS/PAGE, and blotted to nitrocel- lulose. After blocking with 5% skim milk solution, membranes were incubated with rabbit polyclonal anti-HSF1, (Santa Cruz Biotechnology), antibodies, or monoclonal anti-COX-2 (SC- 19999, Santa Cruz Biotechnology), anti-HSP70 (Stressgene), and anti-b-actin (Sigma) antibodies followed by decoration with PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e31304