UTF1 and Cervical Carcinogenesis Figure 6. Effect of 5-aza on cell proliferation and viability. A) Cell proliferation. Cells were counted every 24 hrs during treatment with 5-AZA. B) Cell viability was measured with MTT test. **, p,0,01; ***, p,0,005 (Mann-Whitney U-Test). Results are representative of 3 independent experiments. doi:10.1371/journal.pone.0042704.g006 Methylation and expression of UTF1 after treatment with 5-aza-29-deoxycytidine After treatment with 5-aza, UTF1 promoter methylation was checked by BP. As shown in Figure 7A, UTF1 promoter appeared to be heavily methylated in all the untreated cell lines tested, with global methylation value being 92% for CaSki, 91 % for SiHa, 79 % for A431 and 76 % for HeLa. Treatment with 5-aza decreased the methylation of the CpG with varying efficacy. In SiHa and CaSki global methylation was decreased of 10% and 8% respectively (Figure 7A, see values in red on graph). On the contrary, the loss of methylation was stronger in A431 and HeLa cell lines (221% and 226%, respectively). At the mRNA level (Figure 7B), treatment with 5-aza inhibited UTF1 expression in 3 of the 4 cell lines, excepted in SiHa were UTF1 mRNA expression remained unchanged. In order to unravel a possible link between UTF1 promoter methylation and expression, we tried to correlate the global methylation and the mRNA levels in the four cell lines. A significant positive correlation was observed between UTF1 mRNA expression and UTF1 mean CpG methylation in A431 and HeLa cell lines (Spearman correlation test, p = 0.0167 and p = 0.0333, respectively, Figure S5), but not in SiHa and CaSki (p = 0.8438 and p = 0.0598, respectively, Figure S5). PLoS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e42704