EMT and Transformation Figure 4. Transformation assays of EPT1 cells. A. EP156T, EPT1 and DU145 cells were cultured in medium containing 0%, 1% and 5% FCS, respectively. B. EP156T and EPT1 cells were cultured in the absence (GF2) or presence (GF+) of growth factors in the medium. C. Assay of anchorage independent growth a. Representative images (106) of colony formation in soft agar at different times as indicated. During the first 3 days, some EPT1 cells kept dividing while EP156T did not. Neither EP156T nor EPT1 cells could grow in soft agar eventually, while many colonies formed in prostate cancer DU145 cells. b. Following 7 days incubation in soft agar cells were quantified as absorbance at OD570 nm as described in methods. doi:10.1371/journal.pone.0003368.g004 medium was changed every 3 days. Twelve weeks later cells were trypsinated and transferred to new plates and grown in EP156T medium with FCS increased to 5%. Proliferation, migration, invasion and wound closure assay For the proliferation assay, 3500 cells in complete medium were added to each well of the 96-well plate. After time course incubation, cell proliferation was assayed using the CellTiter96- HAQueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI, USA). MTS reagent was added directly to culture wells, and following incubation for 3 h at 37uC absorbance was recorded at 490 nm using a 96-well plate reader (Powerwave spectrophotometer). For trans-well migration assays and invasion assays, 1.86105 overnight serum starved cells in serum-free medium were added to the top chambers of 24-well trans-well plates (8 mm size, Cell Biolabs Inc., San Diego, CA, USA) and media containing 10% fetal calf serum (FCS) were added to the bottom chambers. Incubation was for 12 h in the migration assay and 24 h in the invasion assay. Top (nonmigrating) cells were removed, bottom (migrating) cells were stained and absorbance was recorded at 560 nm. All of these assays were done in triplicate and the data are presented as the average absorbance of migrating cells and invading cells. For the scratch wound closure assay, 56105 cells were seeded in 6-wells. A wound was incised 24 h later in the high density area, detached cells were removed and fresh medium was added. Photos were taken of the wounded area at indicated time points. Microarray analysis and real-time quantitative PCR Total RNA was extracted from subconfluent monolayers of cells. One mg of DNAse-treated total RNA was converted into cDNA and next to Cy3-labeled cRNA using the Low RNA Input Linear Amplification Kit PLUS, One-Color kit (Agilent Tech., Santa Clara, CA, USA), according to instructions. The Agilent Human Whole Genome (4644 k) Oligo Microarray with Sure PLoS ONE | www.plosone.org 8 October 2008 | Volume 3 | Issue 10 | e3368