Fig. 3. Smad signaling regulates TGFβ1-mediated N-cadherin expression to promote migration. (A) OKF6 cells were transfected with 100 nM of control siRNA (siCtrl) or S4Si, allowed to recover overnight, serum starved and then treated with TGFβ1 for 24 hours. Smad4, ERK2, E- and N-cadherin protein levels were determined by western blotting. Using real-time PCR, the mRNA levels of Smad4, N-cadherin and GAPDH mRNA expression were determined and normalized to siCtrl TGF-b1 (–) samples. (B) OKF6 cells were transfected with 100 nM of siCtrl, S2Si or S3Si, and then treated with TGFβ1 for 24 hours. Smad2, Smad3, ERK2, E-cadherin and N-cadherin protein expression was determined by western blotting. Using real-time PCR the mRNA levels of Smad2, Smad3, N-cadherin and GAPDH were determined and normalized to siCtrl TGFβ1 (–) samples. (C) OKF6 cells were transfected with siCtrl or S4Si, serum starved and treated with TGFβ1 for 24 hours. The cells were then added to porous polycarbonate filters that had been coated with 5 µg type I collagen, and were allowed to migrate for an additional 24 hours in the presence or absence of TGFβ1. Nonmigratory cells were removed from upper chamber, and migrating cells were counted to determine migration relative to untreated siCtrl-transfected cells. ^*P<0.05, significantly different from the siCtrl, TGF-b1+ samples. The results are representative of at least three independent experiments. (D) OKF6 cells were transfected with siCtrl or S4Si, and 16 hours later the cells were infected as detailed in the Materials and Methods with viral particles containing the pQCXIN empty vector or pQCXIN-Ncad vector at two different concentrations for 8 hours, serum starved for 16 hours, and then treated with TGFβ1 for an additional 24 hours. N-cadherin, E-cadherin, Smad4 and ERK2 (loading control) were examined by western blotting. siRNA-transfected cells were infected with pQCXIN empty vector or pQCXIN-Ncad vector at the lower concentration and then added to porous polycarbonate filters that had been coated with 5 µg type I collagen, and allowed to migrate for 24 hours in the presence or absence of TGFβ1. Nonmigratory cells were removed from the upper chamber, and migrating cells were counted. ^*P<0.05, significantly different from the pQCXIN,siCtrl,TGF-b1(+) samples. ^**P<0.05, significantly different from pQCXIN,S4Si,TGF-b1(+) samples. The results are representative of at least two independent experiments.