Table 2. Expression of cell junction genes in EPT1 cells. Table 2. Cont. Genes Desmosome Fold change EPT1/EP156T p-value DSG2 DSG3 DSC2 DSC3 DSP JUP PKP1 PKP2 PKP3 PPL EVPL GJB2 GJB3 GJB4 GJB5 GJB6 CLDN1 CLDN4 CLDN7 OCLN DST KRT4 KRT5 KRT 13 KRT 14 KRT 15 KRT 16 KRT 17 KRT 23 CAV1 LAMA3 LAMA5 ITGB4 ITGB6 ITGB8 PFN1 Adherens junction CDH1 CDH2 CDH3 CTNNB1 CTNND1 Nectin Gap junction Tight junction Hemidesmosome COL17A1 ITGB4 Focal adhesion 22 293 28 26 29 218 27 26 219 239 22 224 33 2120 23 22 4 220 287 217 236 239 23 22 220 24 237 23 2538 212 232 244 242 2177 241 278 211 23 211 24 211 26 25 22 1.9E-3 7.0E-7 2.7E-7 7.7E-7 1.5E-3 2.9E-7 7.2E-6 4.0E-7 5.0E-9 4.9E-5 1.0E-4 6.9E-8 1.0E-8 6.5E-8 8.0E-4 1.7E-3 2.5E-6 7.7E-7 7.3E-10 3.8E-7 6.0E-10 5.8E-6 2.5E-2 9.6E-4 1.2E-5 4.8E-3 5.1E-8 4.2E-2 3.5E-11 1.2E-4 2.2E-13 8.6E-15 3.3E-4 2.0E-7 5.0E-4 3.2E-5 2.5E-5 9.8E-4 4.3E-5 5.0E-5 2.5E-5 2.6E-6 1.0E-5 2.0E-3 EMT and Transformation Fold change EPT1/EP156T p-value 1.7E-6 4.1E-2 3.3E-5 2.4E-5 4.6E-9 3.3E-5 2.8E-3 1.2E-5 Genes PTK2 PTK2B CFL2 ENAH ITGA11 LPXN PARVA TLN2 22 25 6 2 4 3 2 2 Expression changes of cell junction genes in EPT1 cells according to Agilent Human Whole Genome Oligo Microarrays. Results were presented as fold change compared with EP156T cells. GAPDH expression is given as internal control. Three arrays were done for each cell type. Minus fold change represents underexpressed genes in EPT1 cells. doi:10.1371/journal.pone.0003368.t002 both contact inhibition and increased invasion and migration in EMT are associated with cell to cell junctions [54]. The gene expression of entire modules of cell junctions such as adherens junctions, gap junctions, tight junctions, desmosomes and hemidesmosomes were downregulated almost simultaneously as EPT1 arose from EP156T cells. These cell junction modules not only can contribute to the interaction of epithelial cells and provide a barrier against increased migration and invasion in EMT[2], but also can lead to cell contact inhibition based on cell communication [55]. It has been shown that adherens junctions play an important role in contact inhibition of cell growth [56] and components of gap junctions induce cell contact growth inhibition [57]. One recent publication showed that EMT mediated loss of contact inhibition in hepatocarcinoma cells, and reexpression of E- cadherin restored cell-cell contacts [58]. Work is ongoing to identify the key regulatory mechanisms behind the observed switches of entire functional gene expression modules in EPT1 during EMT and the relationship between loss of contact inhibition and EMT in EPT1 cells. Materials and Methods Antibodies, cell lines and cell culture Mouse monoclonal antibodies were used to detect E-cadherin (BD Transduction Labs., BD Biosciences, San Jose, CA USA, Cat#610181), N-cadherin (BD Transduction Labs. Cat# 610920 and Abcam, Cambrigde, GB, Cat.#ab19348) and b-actin (Abcam Cat# ab11003). The prostate cancer cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC, Rockwell, MD, USA). The derivation and growth conditions for the EP156T cell line have been described [20]. Additional reagents were from Sigma-Aldrich, St. Louis, MO, USA unless indicated otherwise. Generation of EPT1 cells EP156T cells were allowed to grow to full confluence at passage 43 in 6 well plates in EP156T standard medium essentially as described [20]. This medium was modified MCDB153 (Biological Ind. Ltd, Israel) and was supplemented with 1% MEM non- essential amino acids solution, 200 nM hydrocortisone, 10 nM triiodothyronine, 5 mg/ml insulin, 5 mg/ml transferrin, 5 mg/ml sodium selenite, 100 ng/ml testosterone, 5 ng/mL EGF, 50 mg/ mL bovine pituitary extract (Invitrogen), 100 U/ml penicillin, 100 mg/ml streptomycin and 1% fetal calf serum (FCS). The PLoS ONE | www.plosone.org 7 October 2008 | Volume 3 | Issue 10 | e3368