TGFβ modulates the phosphorylation levels of the PTEN C-terminus in PTEN expression in H358 cells, followed by EMT and aberrant cell motility.
(A) H358 cells treated with vehicle or TGFβ were harvested for the analysis of fibronectin and E-cadherin. The relative expression of fibronectin to E-cadherin (F/E ratio) is shown in comparison to that in cells treated with vehicle. Data shown represent the means ± SE. The experiment was repeated three times with similar results. *: p<0.05 (B) A migration assay was performed for the H358 cell line treated with vehicle or TGFβ. Data shown represent the means ± SD. The experiment was repeated three times with similar results. *: p<0.05 The fluorescence intensities of β-catenin (red) in the treated cells were evaluated by using confocal laser scanning microscopy and imaging software. Nuclear staining was performed by Hoechst33342 (blue). The left image in (C) shows cells with no TGFβ stimulation. The right image in (C) shows cells stimulated with TGFβ. The cells incubated with isotype-matched control IgG is shown in the inset in (C). The upper panel in (D) plots the fluorescence intensity of β-catenin (red) and nucleus (blue) over a cross section of cells with no TGFβ stimulation. The lower panel in (D) plots the fluorescence intensity of β-catenin (red) and nucleus (blue) over a cross section of the cells stimulated with TGFβ. These figures are representative of at least three independent experiments. (E, F, and G) Cell extracts were harvested at the indicated periods after treatment with TGFβ for analysis of the levels of total and phosphorylated smad2 (E), Akt473 (F), Akt308 (F), and FAK (G). Results are shown for H358 naïve cells at 0 minutes (lane 1), 5minutes (lane 2), 20minutes (lane 3), 1hour (lane 4), 3hours (lane 5), 6hours (lane 6), 24hours (lane 7), and 48hours (lane 8) after treatment with TGFβ (left in E, F, and G). The ratio of phosphorylated protein to total protein is presented as the intensity level relative to that of H358 naïve cells at 0 minutes (lane 1) after treatment with TGFβ (right in E, F, and G). Data shown represent the means ± SE. The experiment was repeated three times with similar results. *: p<0.05 (H) Cells treated with vehicle or TGFβ for 0 minutes or 24hours were harvested for the analysis of phosphorylated PTEN (pPTEN) and total PTEN. The relative expression of pPTEN to total PTEN (pPTEN/PTEN ratio) is shown in comparison to that in the cells treated with vehicle for 0 minutes. 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”. (I) H358 naïve cells were incubated with vehicle or SB 431542 at 10 μM for one hour before TGFβ treatment. pPTEN/PTEN ratio is shown in comparison to that in cells treated with vehicle. 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”.
