Stem Cell Feature in EMT Cells Figure 4. MiR-200 repressed the self-renewal capacity by regulating Notch1 and Lin28B expression. PC3 PDGF-D cells were transfected with pre-miR-200. 3 days after transfection, cells were split and transfected repeatedly with pre-miR-200 every 3–4 days for 14 days. (A) Photographs of cells are shown: transfection of PC3 PDGF-D cells with pre-miR-200 for 14 days, miR-200b and miR-200c reversed EMT cells to MET cell morphology. (B) The cells were collected for generation of prostaspheres after 14-day transfection with pre-miR-200. MiR-200b and miR-200c reduced the number of prostaspheres. (C) Western Blot showing that Notch1, Lin28B and ZEB1 expressions were down-regulated in PC3 PDGF-D cells transfected with miR-200b and miR-200c. (D) The level of miR-200c significantly decreased in ARCaPM by using real time RT-PCR. (E) Transfection of pre-miR-200c after 6 days reduced the prostasphere-forming ability in ARCaPM cells (Bar:100 mm). (F) Western blot showed that re-expression of miR-200c by transfection of pre-miR-200c increased the E-cadherin expression and repressed expression of ZEB1 and Notch1 in ARCaPM cells, which consistent with the change from mesenchymal to epithelial cell morphology (G). *, p,0.05 compared to control; **, p,0.01 compared to control. (Con: control, 200a: pre-miR- 200a, 200b: pre-miR-200b, 200c: pre-miR-200c, E-cad: E-cadherin). doi:10.1371/journal.pone.0012445.g004 cells co-transfected with 39UTR of Notch1 luciferase plasmid and miR-200b or miR-200c. We found that the activity of 39UTR of Notch1 luciferase was significantly increased in PC3 PDGF-D cells compared with PC3 Neo cells due to lower levels of miR-200b and miR-200c in PC3 PDGF-D cells. Moreover, re-expression of miR- 200b or miR-200c dramatically reduced the activity of 39UTR of Notch1 luciferase in PC3 PDGF-D cells, while re-expression of miR- 200a with one base of seed sequence different from miR-200b and miR-200c had no effects on the activity of 39UTR of Notch1 luciferase (Fig. 5B). These results suggest that the miR-200b and miR-200c regulate the Notch1 expression by binding to 39UTR of Notch1 mRNA. To determine whether Notch1 played an important role in maintaining the stem cells, we treated the PC3 PDGF-D cells with DAPT, a c-secretase inhibitor that inhibits the activation of Notch. Full length Notch1 is usually very low due to its cleavage by many enzymes in these cells; however, we found that the expression of full length Notch1 was slightly more in c-secretase treated cells. We also found that DAPT significantly reduced clonogenic ability of PLoS ONE | www.plosone.org 6 August 2010 | Volume 5 | Issue 8 | e12445