HRG-β1 induces upregulation of the transcription factor Snail and EMT markers in SK-BR-3 cells. (a) The cells were incubated with 25 ng/ml of HRG-β1 for different times after serum starvation. The expressions of Snail, mesenchymal markers including vimentin and fibronectin, and epithelial marker E-cadherin were examined by western blotting. β-actin was evaluated as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference. (b) Immunofluorescence staining of E-cadherin protein. Cells were treated with or without 25 ng/ml of HRG-β1 for 48 h. The green color represents staining of E-cadherin and the blue color represents nuclear DNA staining by DAPI (magnification, ×400). The data were analyzed as the percentages of the control cells (**P < 0.01).