Activation of MMPs by EZH2 Figure 7. Transcriptional silencing of TIMP2 by EZH2. (A) Schematic representation of the promoter regions of the TIMP2 gene. The bent arrow represents the transcription start sites (+1). The lines below the TIMP2 locus represent the regions amplified by PCR using primer sets (set 1, set 2, and set 3 in (B); set 2 in (C) and (D)) designed to amplify regions containing the YY1-binding sites (see Table S3). (B–C) Chromatin immunoprecipitation (ChIP) experiments were performed with chromatin isolated from PC3 (B) and DC145(C) cells using antibodies to EZH2, 3meH3K27, and IgG. Results of our ChIP in both PC3 and DU145 cells are very similar to each other. Shown are ChIP results in DU145 cells. (D) Epigenetic regulation of TIMP2 expression in PC3 cells. ChIP assay was carried out using the antibodies to EZH2, 3meH3K27, MeCP2, RNAP II (Pol II), and IgG control after treatment with DZNep (1 mM), 5-Aza (10 mM), DMSO (control), or EZH2-specific shRNA (shRNA1). (E) qRT–PCR analysis of expression of TIMP2 in PC3 cells after treatment with DZNep (1 mM), 5-Aza (10 mM), or DMSO (control) for 2 days. The experiments were repeated at least twice and results were expressed as a ratio of TIMP2 mRNA to GAPDH mRNA control. * P,0.05, ** p,0.005 (as compared with control (DMSO)). doi:10.1371/journal.pone.0030393.g007 well) were added to the upper chamber, whereas RPMI 1640 medium containing 10% fetal bovine serum was added to the lower chamber and incubated in 5% CO2 at 37uC for 24 h. Nonmigrating cells were gently removed from the upper surface of the filter with a cotton swab; migrating cells attached on the lower surface of the filter were fixed and stained with hematoxylin for counting. For the wound-healing migration assay, DU145 cells were seeded at 70% confluence into 6-well culture dishes and, 24 hours later, infected with EZH2-specific or control shRNA lentivirus. After the cells grew to confluence, scratch wounds were made on the confluent monolayers using sterile pipette tips. Afterward, the culture medium was replaced with fresh, complete medium and cells were incubated at 37uC for 24 h and then fixed and photographed. Cell migration was monitored using Olympus IX71 microscope, cell-migration areas were calculated with DP2-BSW application software (Olympus Imaging America Inc.). Chromatin immunoprecipitation (ChIP) assay Chromatin was isolated from prostate cancer cell lines using the ChIP assay kit (Milipore Inc.) and precipitated with anti-EZH2, anti-3meH3K27, anti-MeCP2, or anti-RNA polymerase II antibodies. Immunoprecipitated DNA was PCR-amplified with the primer sets covering specific regions of the TIMP2 and TIMP3 promoters (Table S3). To amplify the DNA within a linear range of amplification for each gene, PCR was performed with different number of cycles or with a serial dilution of input DNA, and all results shown fall within this range. PCR products were run on agarose gels and visualized by ethidium bromide staining. Zymography assay Gelatin zymography assay was performed using confluent prostate cells, which were maintained in serum-free media for 24 h and concentrated using an Amicon Ultra 10 k (Millipore). Equal amounts of concentrated media were mixed with the SDS sample buffer which did not contain b-mercaptoethanol and loaded onto SDS-PAGE gels containing 0.2% gelatin (Sigma). After electrophoresis, gels were washed three times for 20 min at room temperature with renaturing buffer (2.5% Triton X-100). They were then equilibrated in developing buffer (50 mM Tris- HCl pH 7.5, 0.15 M NaCl, 10 mM CaCl2 and 0.05% NaN3) at room temperature with gentle agitation for 20 min. After removing the developing buffer, the gels were incubated with fresh developing buffer at 37uC for 24 h. Finally, the gels were washed with distilled water, stained with 0.05% Coomassie Brilliant Blue for 1 h, and destained with destaining solution (45% methanol and 10% acetic acid) for 3 h. PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e30393