Journal: Cells

Article Title: EB1 Restricts Breast Cancer Cell Invadopodia Formation and Matrix Proteolysis via FAK

doi: 10.3390/cells10020388

Figure Lengend Snippet: EB1 restricts invadopodia formation via inhibition of FAK activity. ( A , B ) MDA-MB-231 cells were transfected with a control siRNA (siLacZ) or siRNA directed against EB1 (siEB1_2), APC (siAPC_1) and ACF7 (siACF7). Levels of Src, activated Src (p-Y416-Src), FAK and FAK phosphorylated on Tyr397 (p-Y397-FAK) were analyzed by Western blotting using specific antibodies. ( A ) Total Src and p-Y416-Src levels are represented as the mean ± SEM of five independent experiments. The value of each individual experiment is reported. A representative Western blot is shown (left). ( B ) Total FAK and p-Y397-FAK levels are represented as the mean ± SEM of five independent experiments. The value of each individual experiment is reported. A representative Western blot is shown (left). Src was used as a loading control and as a reference to quantify p-Y416-Src, total FAK, and p-Y397-FAK levels. The paired t -test was used to determine significant differences. *** p ≤ 0.001, ** p ≤ 0.01. ( C – G ) MDA-MB-231 cells stably expressing mCherrry, mCherry fused to wild type FAK or mCherry fused to the SH2-binding motif mutant of FAK (FAK Y397F) were transfected with a control siRNA (siLacZ) or a siRNA against EB1 (siEB1) before seeding on fluorescently-labeled gelatin (FITC-gelatin) for 4 h. ( C ) Levels of p-Y397-FAK, mCherry fusion proteins, and EB1 were analyzed by Western blotting (WB) using specific antibodies. α-Tubulin serves as a loading control. ( D ) Representative images of cells fixed and stained with anti-mCherry and anti-Cortactin (not shown) antibodies. The percentage of mCherry positive degrading cells ( E ), the degraded area per cell ( F ), and the number of degradation foci ( G ) are represented as the mean ± SEM of four independent experiments. The mean of each individual experiment is reported. Statistical analysis was performed as described in . Full-length blots are presented in .

Article Snippet: Mouse antibodies against EB1 and Src (L4A1) and rabbit antibody against p-Y416-Src were from Cell Signaling Technology, Danvers, MA, USA.

Techniques: Inhibition, Activity Assay, Transfection, Western Blot, Stable Transfection, Expressing, Binding Assay, Mutagenesis, Labeling, Staining