Activation of MMPs by EZH2 Figure 8. Transcriptional repression of TIMP3 by EZH2 promotes invasion of prostate cancer cells. (A) Western blot analysis of EZH2 and TIMP3 expression in RWPE-1 cells after an infection with the indicated combinations of lentivirus expressing EZH2, TIMP3 and control virus. (B) Invasion assay of RWPE-1 cells after an infection with the indicated combinations of lentivirus expressing EZH2, TIMP3 and control virus. Top: a, EZH2(2)/TIMP3(2); b, EZH2(2)/TIMP3(+); c, EZH2(+)/TIMP3(2); d, EZH2(+)/TIMP3(+). (C) Gelatin zymographic assay for MMP-2 and MMP-9 activity in PC3 and DU145 cells infected with EZH2-specific shRNA (sh1) or control shRNA virus (NT). The upper and lower bands indicate active MMP-9 (92 kDa) and active MMP-2 (62 kDa), respectively. Zymographic band intensities were quantified by densitometry using Quantity One software (Bio-Rad). Each bar represents the mean 6 s.e of three fields counted. * P,0.05, ** p,0.005, *** p,0.001 (as compared with control (NT)). doi:10.1371/journal.pone.0030393.g008 Chemicals The anti-actin antibody (A2066), 5-Aza-29-deoxycytidine (5-Aza, A3656), and EZH2 specific-shRNAs were obtained from Sigma- Aldrich. The anti-TIMP2 (MAB3310), anti-3meH3K27 (07-449), anti-MeCP2 (07-013), anti-RNA polymersase II (05-623) antibodies were purchased from Millipore. The anti-EZH2 antibody (612666) was obtained from BD Biosciences. 3-Deazaneplanocin A (DZNep, 13828) was purchased from Cayman. Anti-TIMP3 (ab39184) was obtained from Abcam. Supporting Information Figure S1 Identification of the downstream target genes of EZH2 associated with metastasis of prostate cancer. (A) RT2 profiler PCR array for Human Tumor Metastasis genes in PC3 cells after an infection with EZH2-specific shRNA or control (NT) lentivirus. The scatter plot of the test vs control samples indicates the validity of the experiment. (B) 21 genes whose expressions are highly affected by EZH2 knockdown are shown (also see Tables S1 and S2). Total RNA was isolated from PC3 cells after an infection with EZH2-specific shRNA or control (NT) lentivirus and processed for PCR array. (C) qRT-PCR analysis to determine endogenous levels of TIMP3 mRNA in the benign prostate cell line RWPE-1 and the malignant prostate cancer cell lines PC3 and DU145. Results were expressed as a ratio of the TIMP3 mRNA to the house keeping gene GAPDH (glyceralde- hyde-3-phosphate dehydrogenase) mRNA control. * P,0.05, ** p,0.005 *** p,0.001 (as compared with control values). (EPS) Table S1 List of genes differentially expressed in DU145 cells after EZH2 knockdown using a Human Tumor Metastasis Real-time PCR Array. (DOC) PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e30393