FIG. 3.

PRC2 downregulation affects CDH1 repression by Snail1 in tumor cell lines. (A) CDH1 mRNA levels were determined by qRT-PCR after coinfecting RWP-1 cells with a retrovirus carrying an siRNA specific for Suz12 and Snail1 or cotransfecting the siRNA for Suz12 and Zeb1 (siSuz12). An siRNA for GFP (siGFP) was used as a control. Cells were selected for 48 h with puromycin after the transfection and before isolation of the RNA. The figure shows the averages ± standard deviations (SD) for three experiments performed in triplicate. With respect to the repression of CDH1 RNA obtained with Snail1 in control cells, the reduced effects of Snail1 in siSuz12 cells were significant, with P values of <0.01 (indicated by asterisks). (B) The activities of the indicated forms of the CDH1 promoter (wild type [WT] and mutated [E1E2E3 mut]) in RWP-1 cells were determined by transient transfection of the pGL3-E-cad (−178/+92) CDH1 promoter, pcDNA-3-Snail1, or pcDNA-3-Zeb1 in the presence of an siRNA against Suz12 or GFP. Averages ± SD (three experiments performed in triplicate) are shown. The asterisks indicate values of repression of the CDH1 promoter that were significantly different in siGFP versus siSuz12 cells, with P values of <0.01. (C) SW-620 cells were infected with a mutant of Ezh2 (H649L), a control plasmid (pBabe), or a retrovirus containing an siRNA specific for Suz12, Ezh2 (siEzh2), or GFP as a control. CDH1 and PTEN mRNA levels were determined by qRT-PCR analysis as described above. The figure shows the averages ± SD for five experiments performed in triplicate. The differences indicated with single asterisks were significant, with P values of <0.01.