EMT and Transformation Figure 1. EPT1 cells have lost contact inhibition. A. Representative growth curves of EP156T and EPT1 cells. Both EP156T and EPT1 cells (36103) were seeded into 96-well plates and cultured in medium containing 1% FCS. Medium was changed every 3 days. At the indicated days (D) MTS reagent was added directly to culture wells and cell proliferation was calculated from the absorbance at 490 nm following incubation for 3 hours at 37uC. Data are presented as the average OD490 in three independent experiments. B. DAPI nuclear staining of EP156T and EPT1 cells at low density (a, c) and high density (b, d). White arrows indicate overlapping nuclei. White bars indicate 10 mm. doi:10.1371/journal.pone.0003368.g001 EMT, such as TWIST2 and ZEB1, were also upregulated in EPT1 cells (Table 1). Entire modules of genes encoding structural components of cell junctions and attachment were changed in EPT1 Cell junctions, especially adherens junctions, tight junctions and desmosomes, are required for the epithelial phenotype and keeping neighbouring epithelial cells strongly attached to each other [29]. The dynamic formation and dissolution of cell-cell junctional complexes is a central process during EMT [3]. Apart from the adherens junctions mentioned above, dissociated tight junctions [30,31] or desmosomes [10] were reported as important features of EMT, respectively. Using Agilent Whole Human Genome Microarray data, we compared the expression patterns of genes involved in adherens junctions, tight junctions and desmosomes between EP156T cells and EPT1 cells. As shown in Table 2, the majority of the examined components of these three groups were expressed at a much lower level in the EPT1 cells than in the parental EP156T cells (Table 2), such as E-cadherin, P-cadherin, b1 and d1 catenins in adherens junctions, claudin 1, 4 and 7 in tight junctions, desmoglein 2 and 3 and desmoplakin 2 and 3 in desmosomes. Very interestingly, it was also revealed that genes encoding other structural components of cell junctions were significantly downregulated in EPT1 compared to EP156T cells (Table 2). Gap junctions connect the cytoplasms of adjacent cells through the end- to-end docking of single-membrane structures. Most of the PLoS ONE | www.plosone.org 3 October 2008 | Volume 3 | Issue 10 | e3368