Figure 1.
TGF-β1 induces epithelial-to-mesenchymal changes in MCF-7 cells.
(A) MCF-7 cells treated with 10 ng/ml TGF-β1 for 24 hrs had a spindle-like morphology and lost intercellular junctions. Magnification, 200×. (B) Western immunoblot analysis of expression of EMT-related proteins. Expression levels of E-cadherin and β-catenin (epithelial markers) in TGF-β1-treated MCF-7 cells were markedly decreased, whereas expression levels of N-cadherin and vimentin (mesenchymal markers) were dramatically increased. TGF-β1: transforming growth factor-β1.
Figure 2.
MCF-7 cells acquire migration and invasion abilities in response to TGF-β1 treatment.
(A) Representative pictures of the scratch wound assay. TGF-β1-treated MCF-7 cells migrated faster than controls. The black lines indicated size of the remaining wound. Data from three independent experiments were analyzed and presented as mean ± SD. (P<0.001). (B) Representative pictures in a transwell migration assay (top).The penetrated cells numbers of MCF-7 treated with TGF-β1 for 12 hrs were significantly increased as compared to controls without TGF-β1 (P<0.001). Representative pictures in a transwell invasion assay (bottom). TGF-β1 treatment led to more MCF-7 cells penetrating matrix than controls. (P = 0.002). The results in both transwell migration and invasion assays were obtained from three independent experiments.
Figure 3.
ELE blocks TGF-β1-induced EMT in MCF-7 cells.
(A) Representative pictures of MCF-7 cells treated with ELE showed that epithelial morphology of cells was maintained even in the presence of TGF-β1. Magnification, 200×. (B) Western immunoblot analysis showed that expression levels of E-cadherin and β-catenin (epithelial markers) and N-cadherin and vimentin (mesenchymal markers) did not differ in cells treated with TGF-β1 and ELE compared to cells with ELE only.
Figure 4.
The TGF-β1-induced migration and invasion abilities of MCF-7 cells were blocked by ELE.
(A) A wound healing assay showed that the migration and invasion abilities of MCF-7 cells with TGF-β1 and ELE did not differ from those of control cells without TGF-β1 (P = 0.68). The black lines showed the size of the remaining wounds. (B) Transwell migration and invasion assays demonstrated that the migration and invasion abilities of MCF-7 cells with TGF-β1 and ELE were comparable with those of cells with ELE alone (P = 0.06, and 0.28, respectively). Data are shown from three independent experiments.
Figure 5.
TGF-β1-mediated upregulation of nuclear transcriptional factors expression was blocked by ELE.
(A) RT-PCR analyses showed that mRNA expression levels of transcriptional factors (SNAI1, SNAI2, TWIST and SIP1) were significantly increased in TGF-β1 treated MCF-7 cells (*: P<0.01, **: P<0.001). (B) Western immunoblot analysis showed that protein expression levels of SNAI1, SNAI2, TWIST and SIP1 were repressed by ELE treatment, especially in SNAI1 and SNAI2.
Figure 6.
ELE facilitates TGF-β1-mediated downregulation of Smad3 expression but inhibits increased Smad3 phosphorylation.
(A) RT-PCR analyses showed that TGF-β1 treatment led to downregulation of Smad3 mRNA expression, and ELE enhanced the reduction in Smad3 expression (*: P<0.01, **: P<0.001). (B) Western immunoblot analysis showed that the ratio of p-Smad3/total Smad3 was increased in a time-dependent manner with 10 ng/ml TGF-β1 (*:P<0.01), although expression levels of total Smad3 were slightly decreased. (C) TGF-β1-induced Smad3 phosphorylation was blocked by ELE and total Smad3 was further decreased in MCF-7 cells treated with TGF-β1 and ELE (*: P<0.01, **: P<0.001).