A FAK inhibitor targeting Tyr397 blocks TGFβ-induced aberrant cell motility, but not TGFβ-induced EMT in H358 cells.
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. (A) 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 1) or TGFβ (lane 2, 3, 4, and 5). The cells were also incubated with vehicle (lane 1 and 2), or FAK inhibitor 14 at 0.1 nM (lane 3), 1 nM (lane 4), and 5 nM (lane 5) (top in A). 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 (bottom in A). 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) 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 (bottom in 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”. (C) 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 (D and E) or FAK inhibitor at 5nM (F and G). The left image in (D and F) shows cells with no TGFβ stimulation. The right image in (D and F) shows cells stimulated with TGFβ. The cells incubated with isotype-matched control IgG is shown in the inset in (D). Each upper panel in (E) and (G) 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 (E) and (G) 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.
