Figure 2

The inhibitory effect of pinocembrin on TGF-β1-induced cell-matrix adhesion, invasion, migration, and epithelial-mesenchymal transition of Y-79 cells involves activation of αvβ3 integrin. (A) Y-79 cells were pretreated with 5 μg/ml anti-αvβ3 monoclonal antibody (mAb) or 80 nM cyclic RGD peptide (cyclo-RGDfV) for 30 min and then stimulated with 10 ng/ml TGF-β1 in the presence or absence of 5 μM pinocembrin for 24 h, and were then subjected to analyses for cell-matrix adhesion as described in Methods. (B) In invasion assay, cells were pretreated with 5 μg/ml anti-αvβ3 monoclonal antibody (mAb) or 80 nM cyclic RGD peptide (cyclo-RGDfV) for 30 min and then stimulated with 10 ng/ml TGF-β1 in the presence or absence of 5 μM pinocembrin for 24 h. Then invasion assay were measured by transwell for 8 h; polycarbonate filters (pore size, 8 μm) were precoated with matrigel. After 8 h incubation, cells on the bottom side of filter were fixed, stained, and counted. (C) In migration assay were measured by transwell for 6 h with polycarbonate filters. After 6 h incubation, cells on the bottom side of filter were fixed, stained, and counted. (D) Cells were pre-treated with TGF-β1 (10 ng/ml) in the absence or presence of 5 μM pinocembrin for 0, 30, 60, 120, 180 min, and then used to the cell-lysate extracts were measured by Western blotting with anti-E-cadherin, anti-N-cadherin, anti-vimentin, and anti-β-actin antibodies as described in Methods. Values represent mean ± SD of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001 compared with the TGF-β1 treatment only).