A FAK inhibitor targeting Tyr397 blocks TGFβ-induced aberrant cell motility, but not TGFβ-induced EMT in H358 cells.

<p>To examine the role of FAK phosphorylation at Tyr397 on TGFβ-induced EMT, Dox-treated H358ON cells expressing Dox-dependent GFP were incubated with vehicle or FAK inhibitor 14 for 24hours before TGFβ treatment. (<b>A</b>) Cell extracts were harvested 24 hours after treatment with TGFβ for analysis of the levels of total and phosphorylated FAK. Dox-treated H358ON cells expressing Dox-dependent GFP were treated with vehicle (lane <b>1</b>) or TGFβ (lane <b>2</b>, <b>3</b>, <b>4</b>, and <b>5</b>). The cells were also incubated with vehicle (lane <b>1</b> and <b>2</b>), or FAK inhibitor 14 at 0.1 nM (lane <b>3</b>), 1 nM (lane <b>4</b>), and 5 nM (lane <b>5</b>) (<b>top</b> in <b>A</b>). The ratio of phosphorylated protein to total protein is presented as the intensity level relative to that in Dox-treated H358ON cells expressing Dox-dependent GFP treated with vehicle (<b>bottom</b> in <b>A</b>). 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 (<b>B</b>) Dox-treated H358ON cells expressing Dox-dependent GFP were treated with vehicle or TGFβ for 48hours in the absence or presence of FAK inhibitor 14 at 5nM, 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 (<b>bottom</b> in <b>B</b>). 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>C</b>) A migration assay was performed for Dox-treated H358ON cells expressing Dox-dependent GFP treated with vehicle or TGFβ for 48hours in the absence or presence of FAK inhibitor 14 at 5nM. Data shown represent the means ± SD. The experiment was repeated three times with similar results. *: p<0.05 To evaluate the effect of FAK inhibitor 14 on localization of β-catenin in Dox-treated H358ON cells expressing Dox-dependent GFP treated with vehicle or TGFβ, the intensities of fluorescence of β-catenin in the cells were evaluated. The cells were treated with vehicle (<b>D</b> and <b>E</b>) or FAK inhibitor at 5nM (<b>F</b> and <b>G</b>). The left image in (<b>D</b> and <b>F</b>) shows cells with no TGFβ stimulation. The right image in (<b>D</b> and <b>F</b>) shows cells stimulated with TGFβ. The cells incubated with isotype-matched control IgG is shown in the inset in (<b>D</b>). Each upper panel in (<b>E</b>) and (<b>G</b>) plots the fluorescence intensity of β-catenin (red) and nucleus (blue) over a cross section of cells with no TGFβ stimulation. Each lower panel in (<b>E</b>) and (<b>G</b>) plots the fluorescence intensity of β-catenin (red) and nucleus (blue) over a cross section of cells stimulated with TGFβ. These figures are representative of at least three independent experiments. </p>