Fig. 4. TGFβ1-mediated N-cadherin expression does not involve ERK1/2, p38 MAPK, Src, PI3-kinase, Rho-associated kinase or Jun, but is blocked by EGF in oral keratinocytes. (A) OKF6 cells were serum starved overnight, pre-treated with DMSO (vehicle control), MEK1/2 inhibitor U0126 (10 µM), p38 MAPK inhibitor SB202190 (SB, 10 µM) or the Src inhibitor PP2 (5 µM) for 30 minutes, and then treated with TGFβ1 for 24 hours. N-cadherin and E-cadherin protein levels were determined by western blot analysis. N-cadherin and GAPDH mRNA expression were determined by real-time PCR and relative expression normalized to DMSO-treated TGF-b1 (–) samples. (B) OKF6 cells were serum starved overnight, pre-treated with DMSO (vehicle control), PI3-kinase inhibitor LY294002 (LY, 10 µM), Rho-associated kinase inhibitor Y27632 (10 µM) or the JNK inhibitor SP600125 (SP, 10 µM) for 30 minutes and then treated with TGFβ1 for 24 hours. N-cadherin and E-cadherin protein levels were determined by western blot analysis. N-cadherin and GAPDH mRNA expression were determined by real time PCR and relative expression normalized to DMSO-treated TGF-b1 (–) samples. The results are representative of three independent experiments. (C) OKF4, OKF6, SCC9, SCC25, SCC68 and UMSCC1 were serum starved overnight and then treated with 20 ng/ml EGF 30 minutes prior to treatment with TGFβ1 (10 ng/ml) for 24 hours. E- and N-cadherin protein expression was determined by western blot analysis. The relative levels of N-cadherin and GAPDH mRNA expression in OKF6 and UMSCC1 cells were determined by real-time PCR, and normalized to untreated samples. The results are representative of at least four independent experiments.