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. 2010 Jul 14;31(9):1567–1575. doi: 10.1093/carcin/bgq147

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© The Author 2010. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Fig. 4. — EZH2 knockdown is not sufficient to restore RUNX3 expression in CRC cell lines. (A) Immunoblots of colon cancer cell lines for EZH2 and RUNX3 with actin as a loading control. For every cell line, the first lane is loaded with negative control siRNA-transfected cell lysate and the second lane with EZH2 siRNA-transfected cell lysate. Despite undetectable levels of EZH2 protein after siRNA-mediated knockdown, RUNX3 protein levels remain unchanged. (B) Chromatin immunoprecipitation analysis of the RUNX3 promoter in DLD1 cells treated with control or EZH2 siRNA. Two different pairs of primers for RUNX3 promoter region were used (chromatin immunoprecipitations 1 and 2). Levels of tri-methylated histone 3 K27 are reduced by siRNA-mediated knockdown of EZH2. (C) Left panel. Quantification of the efficiency of the siRNA-mediated knockdown of EZH2 on mRNA levels. The EZH2 mRNA expression in negative control siRNA-transfected cells is set at 100%. (C) Right panel. Quantitative RT–PCR shows no differences in RUNX3 mRNA expression after EZH2 knockdown. (D) Quantitative RT–PCR analysis of RUNX3 mRNA levels in cells treated with 5-aza-2’-deoxycytidine (5-aza-dC) and combined treatment of 5-aza-dC with trichostatin A (TSA) shows a considerable increase in RUNX3 mRNA level.