Stem Cell Feature in EMT Cells Figure 1. Cells with EMT signatures displayed increased Clonogenic ability. (A) Photographs of cells were shown: PC3 Neo cells displayed rounded epithelial cell shape and PC3 PDGF-D cells exhibited a fibroblastic-type phenotype (left panel, original magnification, 200 X). Western blot analysis showed the expression of transcription repressors associated with EMT, and mesenchymal as well as epithelial markers in PC3 Neo and PC3 PDGF-D cells (right panel, passage 10 to 20). (B) The morphology of ARCaPM and ARCaPE cells was shown (left panel).Western blot analysis indicated the increased ZEB1, vimentin and Notch1 expression and decreased E-cadherin expression in ARCaPM cells compared with ARCaPE (right panel). (C) and (D) Photographs of colonies from PC3 Neo and PC3 PDGF-D or ARCaPM and ARCaPE were shown (left panel). The colony numbers were counted and the data was presented as relative colony numbers of PC3 Neo or ARCaPE designed as 1 (right panel). (E) The colonies grown on soft agar were photographed (left panel, bar, 200 mm). The colony numbers were counted under a phase contrast microscope. Data was presented as colony numbers per field (right panel). **, p,0.01 compared to Neo or ARCaPE cells (N: PC3 Neo cells, D: PC3 PDGF-D cells,p10: passage 10, E-cad: E- cadherin). doi:10.1371/journal.pone.0012445.g001 genes associated with EMT phenotype (Table S1). Interestingly, transcription factors Sox2, Nanog, Oct4 and Lin28B, known to be sufficient to reprogram mouse or human somatic cells to undifferentiated, pluripotent stem cells, were found to be significantly increased in PC3 PDGF-D cells compared to PC3 Neo cells. Concomitantly, downstream targets of these transcrip- tion factors such as Zic2, Zic3, Sall2 and Sall4 were also significantly up-regulated (Table S2). To further confirm the results from microarray gene expression analyses, we have conducted real time RT-PCR and Western blot analysis of selected genes. The results from real time RT-PCR showed 2 to thousand-fold increase in mRNA levels of these transcription factors and their downstream targets in PC3 PDGF-D cells (Fig. 3A). Western blot analysis also demonstrated that the protein expressions of Sox2, Nanog, Stat3, Lin28B and Oct4 were significantly higher in PC3 PDGF-D cells compared with PC3 Neo cells from passage 10 to passage 20 (Fig. 3B). Polycomb group proteins are known to be involved in the regulation of gene repression through chromatin modifications, which is essential for the maintenance of the embryonic and adult PLoS ONE | www.plosone.org 3 August 2010 | Volume 5 | Issue 8 | e12445