EMT and Transformation Figure 3. Migration and invasion of EP156T and EPT1 cells. The migration (A) and invasion (B) abilities of EP156T and EPT1 cells were examined as described in methods. Data are presented as the average OD560 of migrating cells (A) or invading cells (B) in four independent experiments. C. Representative light microscopic images (106) show increased migration into a wound line of EPT1 compared to EP156T cells. doi:10.1371/journal.pone.0003368.g003 term culture showed that EPT1 cells not only grew faster, but also looked more vigorous in medium containing 5% FCS than in the standard EP156T medium, so the concentration of FCS in the medium for EPT1 cells was adjusted from 1% to 5%. One of the most important capabilities of cancer cells is self- sufficiency in growth signals [33]. The complete medium of EP156T and EPT1 cells contained many kinds of growth factors including bovine pituitary extract, insulin and EGF [25]. When the EP156T and EPT1 cells were cultured in basic medium without the above growth factors, most of the cells in both cell lines stopped to proliferate and died after 48 h, while the parallel cells in complete medium grew well (Figure 4B). Anchorage-independent growth is considered a hallmark of transformed cells [34,35]. We tested anchorage-independent growth of EPT1 cells in soft agar. Following 7 days incubation in soft agar, neither EP156T nor EPT1 cells were able to form colonies in contrast to positive control DU145 cells (Figure 4). Although neither formed colonies in soft agar, a difference was observed. Nearly half of EPT1 cells divided during the first 3 days, but then proliferation stopped without colony formation in the end. In contrast, EP156T cells did not divide at all in soft agar. These differences in soft agar growth suggested that EPT1 cells are in a stage of premalignancy following EMT (Figure 4). EMT and transformation assays of EPT1 are summarized in Table 3. PLoS ONE | www.plosone.org 5 October 2008 | Volume 3 | Issue 10 | e3368