FIG. 5.

Snail1 represses PTEN promoter activity. (A) Snail1 represses the PTEN promoter in a dose-dependent manner. The activity of the wild type or the E1E2-mutant PTEN promoter was determined for MDCK cells by transient transfection. When indicated, wild-type or P2A mutant Snail1, wild-type Snail2, or Zeb1 cDNAs were cotransfected at several concentrations: 0.1 (dark gray bars), 1 (light gray bars), and 10 ng (white bars). Black bars correspond to the activity of each promoter in the absence of repressors. The figure shows the averages ± SDs (error bars) of three independent experiments performed in triplicate. Asterisks indicate differences that were significant at a P value of <0.05. (B) Snail1 binds to the PTEN promoter. The GST-Snail1 fusion protein or the GST protein was incubated with a double-stranded ³²P-radiolabeled probe corresponding to the two E boxes of PTEN promoter. Binding experiments were carried out with 150 ng of GST-Snail1 without competitor (−) or competing with an excess of unlabeled wild-type (WT) or mutant oligonucleotide (MUT). Arrow, free probe; arrowhead, specific shifted band.