was considered as hypermethylated, whereas below 20% was considered as hypomethylated [27,29]. White Blood Cell isolation Human white blood mononuclear cells (WBC) were obtained as previously described [30]. Briefly, WBC were isolated from leukocyte-enriched buffy coats by centrifugation on Ficoll- Hypaque. After washings at low centrifugation speed to discard a maximum of platelets, WBC were pelleted, washed with PBS and stored at 280uC until DNA extraction. RNA isolation and PCR analyses One mg of total RNA extracted from cell cultures or LBC samples (NucleoSpin RNAII, Macherey-Nagel), treated with DNase and quantified with a ND-1000 spectrophotometer was reverse transcribed using Superscript II reverse transcriptase (Invitrogen, Gent, Belgium) according to the manufacturer’s instructions. The cDNA were stored at 220uC until use. Quantitative Real Time PCR was performed for UTF1 using Taqman Gene Expression Assay (Applied Biosystems, Carlsbad, CA) primers (Hs00864535_s1) and normalized against GAPDH (Hs99999905_m1). Each sample was analyzed in triplicate. For UTF1 RT-PCR reactions, a set of previously described primers was used: Forward, 59-CGCCGCTACAAGTTCCTTAAA-39 ; Reverse, 59-GGATCTGCTCGTCGAAGGG-39 (annealing tem- perature 55uC, 35 cycles, 76 bp) (Figure S1A) [21]. Housekeeping gene (GAPDH) primers were: Forward, 59-TGATGACATCAA- GAAGGTGGTGAAG-39; Reverse, 59-TCCTTGGAGGC- CATGTGGGCCAT-39 (annealing temperature 60uC, 35 cycles, 240 bp). Samples were run on 1.8% agarose gels containing ethidium bromide and visualized with an UV transilluminator. Tissue microarray construction TMA was constructed as previously described [25]. Briefly, the tissue areas (validated by a histopathologist) used for the construction of TMA were selected on the haematoxylin/eosin slides and on the donor blocks and were sampled using a manual arraying instrument (Beecher Instruments, Sun Prairie, WI). TMA blocks were constructed using 1-mm tissue cores (Alphelys, Plaisir, France). Sections of 5 mm were performed and were coated with paraffin for future use. Immunohistochemistry Sections were deparaffinized, rehydrated in graded alcohols and antigens were retrieved in citrate buffer by microwave heating (5 min 750 W, 15 min 300 W). After endogenous peroxydase inhibition, nonspecific binding sites were blocked with Protein Block Serum-Free solution (Dako, Glostrup, Denmark). Slides were incubated overnight at 4uC with a mouse monoclonal antibody against UTF1 (dilution 1/250, clone 5G10.2, Millipore, Temecula, CA). The specificity of the anti-UTF1 antibody was checked by Western Blot in cells overexpressing the human protein (Figure S1B). Immunoperoxidase staining was performed using the LSAB2 kit (Dako). A negative control was obtained by omitting the primary antibody. Spermatogonia in human testicles were chosen as positive control, as previously reported [21]. Positive cells were visualized using a 3,39-diaminobenzidine (DAB) substrate, and the sections were counterstained with Meyer haematoxylin. A score for the staining was established by determining the intensity and extent of the staining according to an arbitrary scale. For staining intensity, 0 represented samples in which the immunoreactivity was undetectable, whereas 1, 2, and 3 denoted samples with, respectively, a low, moderate, and strong UTF1 and Cervical Carcinogenesis staining. For staining extent, 0, 1, 2, and 3 represented samples in which the immunoreactivity was detectable, respectively, in ,5%, 6% to 25%, 26% to 75% and .75% of the tumor cells. To provide a global score for each case, the results obtained with the two scales were multiplied, yielding a single scale of 0, +1, +2, +3, +4, +6 and +9 [30]. Cell Culture DMEM (Gibco, Carlsbad, CA) and RPMI (Gibco) were supplemented with 10% foetal bovine serum (Gibco), 2 mM L- glutamine (Gibco), 1% Sodium pyruvate (Gibco), 1% non essential amino acids (Gibco), 100 U/ml penicillin G, 100 mg/ml strepto- mycin (Gibco). Cell cultures were passaged every three days The cancer cell lines derived from the anogenital area A431, CaSki, SiHa, and Hela (all from the American Type Culture Collections (ATCC)) were grown in DMEM. The teratocarcinoma NCCIT cell line (from the ATCC, kind gift of Pr Luc Grobet, Laboratory of Animal Histology and Embryology, Faculty of Veterinary Medicine, University of Liege, Belgium) treated or not with retinoic acid (all-trans retinoic acid, 10 mM, 8 days, Sigma, St Louis, MO) in RPMI was used as negative and positive controls for UTF1 expression, respectively [18]. For DNA-methyltransfer- ase inhibition, cells were treated with 5-aza-29-deoxycytidine (Sigma) at 10 mM for 72 hrs, with medium changed every day. Then cells were pelleted and stored at 280uC until analysis. Protein extraction and western blot analysis Cells were lysed in RIPA buffer (Pierce, Rockford, IL) supplemented with 1 mmol/L phenylmethyl sulfonyl fluoride (Sigma), and protease inhibitors (Roche). After quantification (BCA protein assay, Pierce), proteins were separated by electro- phoresis on 4% to 12% NuPAGE polyacrylamide gels (Invitrogen) and transferred onto polyvinylidene difluoride membranes. The membranes were subsequently blocked with 5% skim milk for 30 minutes and incubated overnight at 4uC with anti–b-actin (Sigma), anti-UTF1 (mouse monoclonal clone 5G10.2, Millipore), anti-Oct4A (mouse monoclonal sc-5279, Santa Cruz Biotechnol- ogy, CA [31]) or anti-Sox2 (rabbit monoclonal clone D6D9, Cell Signalling, Danvers, MA). The membranes were then incubated with appropriate secondary antibodies (Amersham Biosciences, Piscataway, NJ). After washing, proteins were detected using an enhanced chemiluminescence system (ECL Plus, Amersham Biosciences). Effect of 5-aza-29-deoxycytidine on cell proliferation and viability For the proliferation assay, cells were seeded in 6-well plates and allowed to attach for 24 hrs. Then the cells were treated or not with 5-aza for 72 hrs as described in the section ‘‘Cell Culture’’. Every 24 hrs, adherent cells were harvested and counted. Experiments were performed twice in triplicate. Cell viability was evaluated using a classical MTT reduction assay (Roche, Vilvoorde, Belgium) according to the manufacturer’s instructions [32]. Immunofluorescence Immunofluorescence was performed as previously reported [18]. Cells were plated in two well Lab-Teck slides for 48 h in DMEM. After washing with 0.5 M Phosphate Buffer Saline 1X (PBS), cells were fixed with 4% paraformaldehyd for 15 minutes at room temperature, and then permeabilized with 0.5% of Triton- X100 for 5 min followed by washings in PBS. Slides were incubated overnight at 4uC with anti-UTF1 antibody (1/600, PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42704