clone 5G10.2, Millipore) or antibody diluents without anti-UTF1 antibody as negative control. Goat-anti mouse labeled with rhodamine (1/500, Invitrogen) was used as secondary antibody. Before mounting, slides were briefly washed with dH2O and cell nuclei stained with 49,6-diamino-2-phenylindole (DAPI, 1/2000, Boehringer Ingelheim, Ingelheim am Rhein, Germany) for 5 min. After mounting slides with Eukitt, pictures were acquired using an epifluorescence microscope (Carl Zeiss Inc., Oberkochen, Ger- many). Data analysis All results are given as mean values 6 SE. Statistical analyses and graphical representations were performed with GraphPad Prism 5 software (San Diego, CA). All results were considered significant when p-values were less than 0.05. Statistical tests used are indicated in Figure legends. Results Methylation status of stem cell genes in normal and cancer tissues of the uterine cervix We first analyzed by microarray the methylation profile of 31 genes (68 CpG) specific for SC in the DNA isolated from 4 normal Ecto and 5 cervical SCC. A mean b-value for each CpG was calculated for the 5 SCC and compared with the mean b-value for the 4 Ecto (Db). We then applied a threshold with a Db.0.2 and p,0.05 (Mann-Whitney U-test) in order to identify hypermethy- lated genes in SCC [27,29]. Values are given in Table S1. Using an unsupervised hierarchical cluster analysis we obtained two groups, one encompassing the 4 Ecto and the other group the 5 SCC (Figure 1). Only 7 genes exhibited a Db.0.2 with p,0.05 (Figure 1): UTF1 (2/2 CpG), FGF7 (1/2 CpG), MSI1 (1/2 CpG), SOX2 (1/2 CpG), SMO (1/2 CpG), DNMT3L (1/1 CpG) and SALL4 (1/1 CpG). We then focused our experimentations on UTF1, since the two CpG of this gene were the most hypermethylated in SCC (Db = 0.685 and 0.545, p,0.05 for each CpG). This gene is composed of two exons separated by a short intron. The Oct4/Sox2 enhancer is located upstream the 39 sequence, (Figure S2). The two CpG analyzed in our microarray are located in a CpG island covering the promoter region, the transcription start signal and the first exon (red triangles, Figure S2). Methylation status of UTF1 promoter in DNA isolated from liquid-based cytological samples We then used direct BP analysis to confirm the methylation status for the CpG located in UTF1 promoter. We set a cut-off value of 20%, meaning that CpG methylation above this limit was considered as hypermethylated, whereas below 20% was consid- ered as hypomethylated [27,29]. We first validated our results by analyzing the same samples that were used for microarray analysis. We observed that UTF1 promoter was hypomethylated in normal Ecto, whereas the same sequence was demonstrated as totally hypermethylated in SCC. (Figure S3 and Table S2). To confirm UTF1 promoter methylation signature, we performed direct BP on DNA isolated from liquid-based cytological (LBC) of four groups of samples: Ecto, LSIL, HSIL and SCC. We applied the same 20% cut-off as for DNA extracted from frozen tissues. In Ecto and LSIL, CpG were mostly hypomethylated and there was no difference in CpG methylation levels between these two groups (Figure 2A, 2B and Table S3, p = NS). There was a strong hypermethylation of UTF1 promoter in HSIL and all the CpG in SCC samples were considered as totally hypermethylated (Figure 2A and 2B). Moreover, in SCC, all the CpG were UTF1 and Cervical Carcinogenesis significantly much more methylated than in Ecto and LSIL samples (Table S3, p,0.001, Ecto and LSIL vs SCC). Interest- ingly, CpG methylation levels in SCC were also significantly higher compared to HSIL (Table S3, for each CpG analyzed, p at least ,0.05, SCC vs HSIL). Because LBC samples usually consist of heterogeneous mixtures of abnormal and normal squamous cells, as well as of inflammatory cells [33], we also analyzed the methylation status of UTF1 promoter in DNA of 10 samples of white blood cells (WBC). As shown in Figure 3 and Table S4, in all WBC conditions, the promoter of UTF1 appeared to be totally hypomethylated, with CpG methylation values ranging from 3 to 11 %, suggesting that UTF1 methylation profile observed in LBC samples is not related to inflammatory cells. Expression profile of UTF1 during cervical carcinogenesis We then analyzed the expression of UTF1 mRNA by quantitative RT-PCR in the same LBC samples. As shown in Figure 4A, in Ecto and LSIL, UTF1 mRNA were similar (p = NS). Its expression increased in HSIL but not significantly. On the contrary, SCC expressed significantly more UTF1 mRNA than in Ecto and LSIL (more than 6 fold, p,0.05 and p,0.01, respectively). A significant positive correlation was observed between UTF1 mRNA expression and UTF1 mean CpG methylation (Spearman correlation test, p = 0.0003, Figure S4). UTF1 expression was then analyzed by immunohistochemistry on a TMA dedicated to cervical carcinogenesis (18 Ecto, 11 LSIL, 18 HSIL and 19 SCC) with a previously described mouse monoclonal antibody [19,21]. Among the 18 Ecto, Weak UTF1 staining was observed in the cell nuclei of the basal layer and of the epithelium in 90% of the samples, whereas it was not expressed by the remaining 10% samples (Figure 4B). The staining for UTF1 in LSIL was localized in the first third of the epithelium in all the samples tested and was similar to that observed in Ecto (Figure 4B and 4C, p = NS, LSIL vs Ecto). In contrast, immunoreactivity of UTF1 was much stronger and found in all cells of 92% of the HSIL samples, whereas it was not expressed by only one case (Figure 4B and 4C, p,0.05 vs LSIL). In SCC, staining of UTF1 was significantly stronger than in Ecto and LSIL in 75% of the samples (14 cases) (Figure 4B and 4C, p,0.01 vs Ecto and LSIL) and was expressed by all the cells within tumor samples. Expression of UTF1 and its regulators Oct4A and Sox2 in cancer cell lines We next studied the expression of UTF1 mRNA and protein in several cell lines derived from the anogenital area (A431, SiHa, CaSki and HeLa). The expression of UTF1 mRNA was compared to that of the NCCIT carcinoma cell line which express high level of UTF1 while it was very low when these cells are treated with retinoic acid (RA) [18]. As shown in Figure 5A, in cancer cell lines, except for CasKi cells, UTF1 mRNA was more expressed, than in NCCIT differentiated with RA (p,0.001 vs NCCIT+RA). The same profile of UTF1 mRNA expression in cell lines was confirmed by end-point RT-PCR (Figure S1A), using previously published primers [21]. At the protein level, UTF1 was found in all cell lines, except that it was detected in CaSki and not in HeLa cells, suggesting post- transcriptional regulation of UTF1 expression (Figure 5B). The specificity of the mouse monoclonal antibody was validated by overexpressing UTF1 with a plasmid containing UTF1 human cDNA in SiHa and A431 cells (Figure S1B). We also analyzed the expression of the known regulators of UTF1 expression and showed that neither Oct4 nor Sox2 were expressed in any cell lines (Figure 5B). The nuclear localization of UTF1 was confirmed by immunofluorescence in the cell lines (Figure 5C). PLoS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42704