Table 3. Summary of EMT and transformation assays of EPT1 cells. EMT In vitro functional markers Elongation of cell shape Increased scattering Increased migration Increased invasion Increased mesenchymal markers N-cadherin/Vimentin/Fibronectin/Integrin Decreased epithelial markers E-cadherin/Desmoplakin/Cytokeratin/Occludin Transformation Loss of contact inhibition Chromosomal instability Serum independent growth Self-sufficiency in growth signals Anchorage independent growth doi:10.1371/journal.pone.0003368.t003 Yes or No Yes Yes Yes Yes Yes Yes Yes No No No No Yes or No Print Technology (Agilent Technologies, Inc., Palo Alto, CA) was used to analyze samples in the present study. The hybridization and features extraction were described in [59]. Significant Analysis of Microarray (SAM) using the significantly up- or downregulated genes in EPT1 cells were performed in J-Express (www.molmine. com) [60]. Annotated microarray data were uploaded in the BASE database and formatted and exported to ArrayExpress at the European Bioinformatics Institute (http://www.ebi.ac.uk/ arrayexpress (Accession number: E-BASE-8)) in agreement with the MIAME guidelines. Differentially expressed genes in micro- array data were confirmed by real-time quantitative reverse transcription PCR (qPCR) using TaqMan assays (Applied Biosystems, Foster City, CA, USA) and mean quantitative values were normalized according to ACTB expression as described [59]. Indirect immunofluorescence and Western blot assays Indirect immunofluorescence (IF) and western blot (WB) analyses were done essentially as previously described [61]. For IF cells were grown on 10 mm Assistant glass coverslips in 24 well plates, then washed with PBS, fixed (4% fresh paraformaldehyde in PBS for 20 min. at room temperature), permeabilized (0.5% Triton X-100 for 10 min.), blocked (100 mM glycin for 10 min) and stored (100% methanol at 4uC) with PBS wash between each step. Following blocking with 0.5% BSA/PBS for 15 min. primary mouse monoclonal antibodies were added overnight at 4uC at indicated dilutions in 0.5% BSA/PBS. The FITC-labelled secondary anti-mouse Ig (SouthernBiotech, Birmingham, AL, USA, Cat# 4050-02) was added for 1 h at room temperature in 0.5% BSA/PBS. Coverslips were mounted in SlowFade with DAPI (Molecular Probes, Invitrogen, Carlsbad, CA, USA) on glass slides and analysed using Leica confocal microscopy. For WB analysis cells were lysed in 200 mM Tris.Cl pH 6.8, 13% ultrapure glycerol, 3.2% b-mercaptoethanol, 40 g/l SDS and Protease Inhibitor Cocktail Set I diluted 1:100 (Calbiochem, Cat# 535142). Protein concentrations were measured using the BCA protein Assay kit (Pierce, Rockford, IL, USA, Cat# 23227), and 20 mg protein lysates were separated by 10% polyacrylamide gel EMT and Transformation (Biorad Labs., Hercules, CA, USA, Cat#161-1173) SDS electro- phoresis followed by blotting to PVDF membranes (GE Health- care Life Sciences, Uppsala, Sweden, Cat# RPN1416F). Primary mouse monoclonal antibodies and HRP-labelled secondary antibodies (GE Healthcare, Cat.# NA931) were used at the indicated concentrations and the bands detected using enhanced chemiluminescence (GE Healthcare, Cat# RPN2132) and the Biorax Fluor-s multiImager. Molecular weight markers used were ECL DualVue Marker (GE Healthcare, RPN810) and Magic- Mark XP Western Protein Standards (Invitrogen, Cat# LC5602). Serum independent, growth factors independent assays For the serum independent assay, cells were cultured in medium with 0%, 1% and 5% FCS, respectively. For the growth factors independent assay, cells were cultured in basic MCDB medium and complete medium, respectively [20]. The proliferation of each group was measured 48 h later as described above. Anchorage-independent growth assay The anchorage independent growth was examined in soft agar. 50 ml of base agar matrix (Cell Biolabs; CytoSelectTM Cell, Colorimetric kit) was added in the bottom of each well of a 96-well plate. When the agar was solid, 75 ml of cell suspension/soft agar matrix containing 3000 cells was layered on top followed by 50 ml of 26 complete medium. After 7 days of incubation, the agar matrix was solubilized and cells stained and absorbance was recorded at 570 nm. Data show the quantification of proliferation of EPT1 cells, EP156T cells and positive control DU145 cells in the soft agar assay. Cytogenetic analysis Prior to harvesting, the cells where treated with 20 ng/ml colcemid for 25 min at 37uC followed by trypsination. The cell suspension was washed once before exposing the cells to prewarmed (37uC) hypotonic solution (0.22% (v/v) NaCl, 0.3 mM KH2PO4, 80 mM Na2HPO4, pH 8.0). Cells were pelleted and re-suspended in 4 ml fresh fixative (3:1 methanol:a- cetic acid) which was repeated four times before storing the cell suspension at 220uC. G-banding was performed by the standard Giemsa staining procedure and metaphase spreads were analysed. The karyotype was described according to ISCN 2005. FISH (fluorescent in situ hybridization) analyses using probes (CEN20, TEL12q, LSI MLL) from Vvsis (Abbott Molecular–Vysis, Des Plaines, IL; Abbott Diagnostics, Maidenhead, UK) were per- formed according to the manufacturer’s instruction. Supporting Information Figure S1 Hierarchical clustering analysis of differentially expressed genes among DU145, PC3, EP156T and EPT1 cells. In total 1858 genes differed more than 3 fold between EPT1 and EP156T cells. The red and the blue represent low expression and high expression, respectively. Found at: doi:10.1371/journal.pone.0003368.s001 (4.20 MB TIF) Figure S2 G-banding of metaphases of the EP156T cells (A) showed few chromosomal aberrations compared to prostate cancer cell lines. A marker chromosome was found in all cells (der(20), red arrow), but diverging clonal evolution as often seen in cell lines was also seen in the EP156T cells. One subclone had trisomy 13 (blue arrow), another loss of chromosome 8 and 20 and gain of chromosome 2, whereas others showed different marker chromo- somes. The composite karyotype can be described as 46– 48,XY,+2[2],28[3],+13[4],220[2],+der(20)[10],+mar[3][cp10]. G- PLoS ONE | www.plosone.org 9 October 2008 | Volume 3 | Issue 10 | e3368