Mutation of phosphorylation sites in the PTEN C-terminus blocks TGFβ-induced EMT and aberrance cell motility in H358 cells.

<p>(<b>A</b>) H358ON cells expressing Dox-dependent GFP, GFP-PTENWt, or GFP-PTEN4A were incubated with vehicle or Dox for 24hours before TGFβ treatment. The cells were then treated with vehicle or TGFβ for a further 24hours in the absence or presence of Dox. The cells were harvested for the analysis of pPTEN (<b>top</b> panel), total PTEN (<b>middle</b> panel) and β-actin (<b>bottom</b> panel) by western blotting. A representative blot from three independent experiments is shown. (<b>B</b>) By using confocal laser scanning microscopy, the localization of GFP fluorescence in H358ON cells expressing Dox-treated GFP (<b>left panel</b>), GFP-PTENWt (<b>middle panel</b>) and GFP-PTEN4A (<b>right panel</b>) was evaluated. (<b>C</b>) The intensity levels of GFP fluorescence in both the cytoplasm and the nucleus were also quantified, by Imaging software. The fluorescence intensity was expressed as the nucleus/cytoplasm ratio for each sample. Data shown represent the means ± SEM from three independent experiments. *: p<0.05 N.S. indicates “not significant”. (<b>D</b>) H358ON cells expressing Dox-dependent GFP, GFP-PTENWt, or GFP-PTEN4A were treated with vehicle or TGFβ for 48hours in the absence or presence of Dox, and then harvested for the analysis of fibronectin, E-cadherin, and β-actin by western blotting. The F/E ratio is shown in comparison to that in cells treated with vehicle in the absence of Dox. A representative blot from three independent experiments is shown. Data shown represent the means ± SE. The experiment was repeated three times with similar results. *: p<0.05 N.S. indicates “not significant”. (<b>E</b>) A migration assay was performed for H358ON cells expressing Dox-dependent GFP, GFP-PTENWt, or GFP-PTEN4A in the absence or presence of Dox and/or TGFβ stimulation. Data shown represent the means ± SD. The experiment was repeated three times with similar results. *: p<0.05 N.S. indicates “not significant”.</p>