β3 integrin blockade or silencing inhibits invadopodia formation by H157 NSCLC cells.

<p><b>(A)</b> Flow cytometry and Western blot analysis of β3 integrin expression in control and TGF-β treated H157 cells before and after β3 integrin silencing with a specific shRNA. β-Actin was used as Western blot loading control. <b>(B)</b> Quantification of the percentage of H157 NSCLC cells with gelatin-degrading invadopodia after β3 integrin blockage by preincubation with anti-β3 integrin mAbs (1 μg of antibody for 2 hours before the experiment). Cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181579#pone.0181579.g001" target="_blank">Fig 1</a>. Isotypic non-specific IgG treatment was included as control. Data represent mean ± SEM of four different experiments in which at least three fields and a total of 100 cells per experiment were analysed. For statistical analyses a one-way ANOVA followed by post-hoc Tukey´s tests for multiple comparisons was used. ** p< 0.01 and *** p< 0.001. Panels show representative microphotographs of gelatin-degraded areas obtained in each experimental condition. Scale bars 23 μm. Red asterisks indicate degradation sites. <b>(C)</b> Quantification of the percentage of cells with gelatin-degrading invadopodia in H157 cells expressing or not β3 integrin and treated or not with TGF-β. Cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181579#pone.0181579.g001" target="_blank">Fig 1</a>. Data represent mean ± SEM of four different experiments in which at least three fields and a total of 100 cells per experiment were analysed. For statistical analysis one-way ANOVA followed by post-hoc Tukey´s tests for multiple comparisons was used. ** p< 0.01, *** p< 0.001. Panels show representative microphotographs of gelatin-degraded areas obtained in each experimental condition. Scale bars 23 μm. Red asterisks indicate degradation sites. <b>(D)</b> 3D reconstructions obtained from confocal-laser microscopy images captured from H157 cells grown in DQ-collagen matrices for 72 hours. Actin (red) and DQ-collagen degradation areas (green) are shown in wild type and β3 integrin-silenced cells treated or not with TGF-β for 5 days. Scale bar 10 μm and 7μm, as indicated. White asterisks indicate examples of collagen degradation sites associated with actin protrusion tips, whereas white arrowheads point to actin puncta without associated degradation. Big white arrow signals cell orientation. The white boxes show a magnified image of one selected. <b>(E)</b> Western blot analysis and signal quantification by densitometry of total and phosphorylated Smad2/3 levels in H157 cells and β3 integrin silenced cells treated, or not, with TGF-β. β-Actin was used as loading control.