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Figure 5 | Breast Cancer Research

Figure 5

From: Therapeutic targeting of the focal adhesion complex prevents oncogenic TGF-β signaling and metastasis

Figure 5

Focal adhesion kinase (FAK) is critical for TGF-β stimulation of invasion in malignant mammary epithelial cells (MECs). (a) Fluorescent-conjugated phalloidin staining of control (i.e., scram) and FAK-deficient (shFAK) 4T1 cells indicated that FAK deficiency (shFAK) preserved cortical actin staining at cell-cell borders with TGF-β1 (5 ng/ml) stimulation. Data are representative images from a single experiment that was repeated twice. (b) Control (i.e., scram) and FAK-deficient (shFAK) 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for varying times, as indicated. Afterward, cell extracts or supernatants were immunoblotted with antibodies against E-cadherin (E-cad) or plasminogen activator inhibitor-1 (PAI-1), respectively. Immunoblotting for β-actin served as a loading control. Data are representative images from a single experiment that was repeated twice and show that FAK deficiency inhibited epithelial-mesenchymal transition (EMT) stimulated by TGF-β. (c) Control (i.e., scram) and FAK-depleted (shFAK) 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 24 hours. Afterward, the downregulation of the epithelial markers E-Cad and cytokeratin-19 (CK19), and the upregulation of the mesenchymal markers, PAI-1, matrix metalloproteinase 9 (MMP9), cyclooxygenase-2 (Cox2), and β3 integrin (Beta3) were analyzed with semiquantitative real-time PCR. Data are expressed as the mean fold change (± SEM) in gene expression relative to unstimulated cells observed in at least three independent experiments (*P < 0.05). (d) FAK deficiency specifically inhibited cell invasion induced by TGF-β. Data are the mean (± SEM) invasion of each 4T1 cell line normalized to serum-stimulated invasion observed in three independent experiments completed in triplicate. (*P < 0.05; **P < 0.005). (e) FAK deficiency reversed TβR-II-mediated cellular invasion. Data are expressed as the mean (± SEM) invasion of each 4T1 cell line relative to its unstimulated counterpart (NS) observed in three independent experiments completed in triplicate. (*P < 0.05; **P < 0.001).

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