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. Author manuscript; available in PMC: 2012 Mar 2.
Published in final edited form as: Nat Cell Biol. 2010 Oct 10;12(11):1108–1114. doi: 10.1038/ncb2116

Figure 3.

Figure 3

The effect of Thr 350 phosphorylation on EZH2-mediated repression of its target-genes. (a) Hierarchical clustering of 6,450 genes (represented by 10,276 probe-sets) that exhibited expression differences in LNCaP cells. Lanes 1–4; cells were transfected with EZH2-specific siRNA (EZ4) and vectors expressing siRNA-resistant EZH2 or a siRNA-resistant EZH2T350A mutant, as indicated. Lanes 5–7; cells were treated with a CDK inhibitor or HDAC inhibitor. Gene profiling data from cells transfected with the indicated siRNA and vectors (lanes 2, 3 and 4) were normalized to that in cells transfected with control siRNA (lane 1), and the data from drug-treatment experiments (lanes 6 and 7) were normalized to vehicle (DMSO) treatment (lane 5). Red and green represent upregulation and downregulation, respectively, as indicated in the scale at the top. Wild-type EZH2 and EZH2T350A mutant proteins were expressed at comparable levels (Supplementary Information, Fig. S3b). Experiments were performed in duplicate (n = 2). (b) LNCaP cells were transfected with EZ4 siRNA or plasmids expressing Myc-tagged, siRNA-resistant EZH2 or Myc-tagged, siRNA-resistant EZH2T350A, as indicated (empty vectors were used as a control). At 60 h after transfection, expression of HOXA9 (left) and DAB2IP (right) were analysed by real-time RT–PCR. Asterisks indicate P < 0.01. Experiments were performed in triplicate (n = 3). (c) Prostate cancer cells were transfected with plasmids expressing v5–CDK2 and v5–cyclin E (DU145 cells, left), v5–CDK1 and v5–cyclin B1 (PC-3 cells, right), or an empty control vector, in combination with control or EZH2 siRNA. At 72 h after transfection, expression of HOXA9 was evaluated by real-time RT-PCR. Asterisk indicates P < 0.05, and double asterisks indicate P < 0.01. Experiments were done in triplicate (n = 3). Western blots (bottom) were used to identify expression of the indicated proteins. (d) DU145 cells were transfected with siRNAs against CDK1, CDK2 and EZH2 as indicated. At 72 h after transfection, expression of HOXA9 was evaluated by real-time RT-PCR. Asterisks indicate P < 0.01. Experiments were carried out in triplicate (n = 3). Western blots (bottom) were used to identify expression of the indicated proteins. Uncropped images of blots are shown in Supplementary Information, Fig. S6a.