PCR and western blot analysis of the expression of SMAD4 and genes related to EMT in GH3 cells. A: The mRNA levels of SMAD4 and genes related to EMT closely correlated with transfection time. B: The protein levels of SMAD4, E-CAD, N-CAD, and Vimentin in GH3 cells after overexpression of SMAD4. The levels of SMAD4 and Vimentin peaked after 72 h of transfection. C: Statistical analysis of western blot data.