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

(A) 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 (top panel), total PTEN (middle panel) and β-actin (bottom panel) by western blotting. A representative blot from three independent experiments is shown. (B) By using confocal laser scanning microscopy, the localization of GFP fluorescence in H358ON cells expressing Dox-treated GFP (left panel), GFP-PTENWt (middle panel) and GFP-PTEN4A (right panel) was evaluated. (C) 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”. (D) 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”. (E) 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”.