Fig. 7

β1-integrin is implicated in the epithelial-mesenchymal transition process induced by cancer-associated fibroblasts in MCF-7R cells. (a) Equal amounts of total cell protein were separated by SDS-PAGE and subsequently probed with antibodies against E-cadherin, fibronectin and vimentin. (b) Localization and expression of E-cadherin and vimentin (red) in MCF-7, MCF-7R, and cancer-associated fibroblast (CAF) cell lines were revealed by immunofluorescence staining. The nucleus was stained blue with DAPI. Original magnification: ×200. (c) Levels of membrane E-cadherin were detected by Western blotting using specific antibodies. Na⁺/K⁺-ATPase was used for normalization. (d) Cells were cultured in CAF-conditioned medium (CM) or normal medium for 48 hours, and the expression of E-cadherin, fibronectin and vimentin was then determined by Western blotting. (e) β1-integrin expression in MCF-7R cells was blocked by lentivirus vector transfection (left panel). Cells were left untreated (control; Ctrl) or treated with CM for 48 hours, or pretreated with the PI3K inhibitor Wortmannin (10 μM; WM) or the MAPK/ERK inhibitor U0126 (10 μM) prior to CM treatment, and then cell lysates were probed with antibodies against fibronectin and vimentin (right panel). (f) Cell migration of MCF-7R cells with indicated pretreatments was measured through Transwell assays. Each experiment was repeated at least three times. Western blotting results are shown as fold-changes in optical density compared with the control, and normalized to β-actin. *P < 0.05. ITGB1, β1-integrin; sh/ITGB1, MCF-7R cells infected with a lentivirus vector targeting β1-integrin; sh/Vec, MCF-7R cells infected with negative control lentivirus