Activation of MMPs by EZH2 Figure 6. Expression of TIMPs is differently regulated by EZH2. (A–C) qRT–PCR analysis of expression of TIMP genes (A, TIMP1; B, TIMP2; C, TIMP4) in the prostate cancer cells (LNCaP, DU145, and PC3) after an infection with EZH2-specific shRNA (sh1) or control shRNA lentivirus (NT). * P,0.05, ** p,0.005 (as compared with control (NT)). (D–F) qRT–PCR analysis of expression of TIMP genes (D, TIMP1; E, TIMP2; F, TIMP4) in the benign prostate cell line RWPE-1 after an infection with EZH2 overexpression lentivirus (WT or H689A) or control virus for 4 days. Results were expressed as a ratio of TIMP mRNAs to GAPDH mRNA control. * P,0.05, ** p,0.005 (compared with control (GFP)). doi:10.1371/journal.pone.0030393.g006 BioChain Institute, Inc. Prostate cancer cells were grown on chamber slides (Nunc) and infected with either control or EZH2- specific shRNA lentivirus. After cultivating for 2–4 days, the cells were fixed with cold methanol and permeabilized with buffer containing 1% BSA, 10% normal goat serum, 0.3 M glycine, and 0.1% PBS-Tween for 1 h. The resulting cells were incubated with anti-TIMP3 and anti-EZH2 antibodies at 4uC overnight. After washing, they were incubated with Alexa FluorH 594 goat anti-rabbit IgG and Alexa FluorH 488 goat anti-mouse IgG at a 1/1000 dilution for 1 h. After mounting with mounting medium containing DAPI (Vector Labs, H-1200), the cells were photographed using a fluorescence microscope (Olympus Imaging America Inc, IX71 microscope). Cell invasion and migration assays Cell invasion assay was performed using the cell invasion kit (Transwell Boyden’s chamber with TranswellH Permeable Support Inserts Coated with CultrexH BME (basement membrane extract), Corning Costar) according to the manufacture’s instruction. In brief, serum-starved RWPE-1, DU145, or PC3 cells (26105 cells/ PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e30393