Stem Cell Feature in EMT Cells Figure 2. Prostaspheres were increased in cells having EMT phenotype. (A) Prostaspheres from PC3 Neo and PC3 PDGF-D cells were photographed and shown (bar, 100 mm). (B) The numbers of prostaspheres were counted under microscope. (C) Prostaspheres were photographed and the size of prostaspheres in diameter was measured using software image analysis program Scion Image. (D) The prostaspheres (P1) were collected and re-plated at a density of one cell per well in 96-well plate with ultra low attachment. After 12 days of incubation, the numbers of prostaspheres (P2) were counted under a phase contrast microscope. (E) The numbers of prostaspheres from ARCaPE and ARCaPM cells were counted under microscope. (F) Prostaspheres from ARCaPE and ARCaPM cells were photographed and shown (bar, 100 mm). **, p,0.01 compared to Neo or ARCaPE cells (Neo: PC3 Neo cells, PDGF-D: PC3 PDGF-D cells). doi:10.1371/journal.pone.0012445.g002 stem cells [32,33]. Polycomb repressive complex 2 (PRC2) contains Suz12, EZH2, EED and RbAp subunits and functions in the embryonic and adult stem cells to repress developmental genes that are preferentially activated during differentiation. Moreover, further studies have shown that PRC2 target genes are co-occupied by stem cell regulators such as Oct4, Sox2 and Nanog [34]. During the data analysis of our microarray data, we found that the levels of EZH2 and Suz12a mRNA were increased in PC3 PDGF-D cells compared to PC3 Neo cells (Table S2), which were further confirmed by real time RT-PCR (Fig. 3C). In addition, the protein levels of EZH2 was significantly up-regulated in PC3 PDGF-D cells relative to PC3 Neo cells (Fig. 3D) and these results clearly suggest that the EMT-type characteristics of PC3 PDGF-D cells are consistent with the signatures of stem cells or cancer stem-like cells. Notch signaling has been shown to play important roles in pluripotency and self-renewal capacity of both embryonic and adult stem cells [35,36]. The results from our microarray data showed that mRNA levels of Notch, Notch ligands, delta-like, Jagged as well as Notch downstream targets such as Hes and Hey were significantly higher in PC3 PDGF-D cells compared to PC3 Neo cells (Table S2). Real time RT-PCR showed 2 to 350-fold increase in mRNA levels of Notch signaling genes (Fig. 3E), which were further confirmed by Western blot analysis as shown in Fig. 3F. Likewise, we also found significantly increased level of Notch1 expression in ARCaPM cells compared to ARCaPE cells (Fig. 1B, right panel). MiR-200b and miR-200c expression linked cancer stem cell signatures with EMT phenotype In our previous studies, we found significant down-regulation of miR-200 family in PC3 PDGF-D cells with EMT phenotype [28], which was consistent with our miRNA microarray data (Table S3). To reveal whether expressions of miR-200 family are associated with stem cell signatures, PC3 PDGF-D cells were transfected with miR-200a, miR-200b and miR-200c. We found that transfection of miR-200b and miR-200c induced reversal of EMT to MET phenotype (Fig. 4A). More importantly, re-expression of miR- 200b and miR-200c significantly inhibited the prostasphere- forming ability of PC3 PDGF-D cells (Fig. 4B). Moreover, the size of prostaspheres was also reduced in PC3 PDGF-D cells transfected with miR-200b and miR-200c compared to transfec- tion with control miRNA (Fig. S2A and S2B). However, transfection of PC3 PDGF-D cells with miR-200a had no effect on prostasphere-forming ability but increased the size of prosta- spheres compared to control (Fig. 4B, Fig. S2A and Fig. S2B). PLoS ONE | www.plosone.org 4 August 2010 | Volume 5 | Issue 8 | e12445