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Fig. 2. TGFβ1 increases migration of oral keratinocytes by upregulating N-cadherin expression. (A) Equal numbers of cells of the different cell lines were plated, serum starved for 24 hours and then allowed to condition the medium for an additional 24 hours. The conditioned medium was analyzed for total TGFβ1 by ELISA, and cell lysates processed for TGFβ1 and GAPDH mRNA expression by using real-time PCR and normalized to the levels present in OKF4 cells. *P<0.05, significantly different from the OKF4 levels; **P<0.005, significantly different from the OKF4 levels. (B) Oral keratinocytes and OSCC cells were serum-starved overnight, treated with TGFβ1 for 24 hours and the effect on E- and N-cadherin expression was determined by western blot analysis. Using real-time PCR, expression levels of E-cadherin, N-cadherin and GAPDH mRNA were determined and normalized to untreated samples. (C) SCC25 cells were transfected with 100 nM of control siRNA (siCtrl) or siRNA targeting TβRI (siTβRI); then TβRI, ERK2, N-cadherin and E-cadherin protein levels were determined 72 hours later by western blot analysis. Using real-time PCR, the relative expression of TβRI, E-cadherin, N-cadherin and GAPDH was determined and normalized to siCtrl-transfected cells. (D) SCC25 cells were serum starved overnight and then treated with DMSO (vehicle control) or with the TβRI inhibitor (TbRi, 10 µM) for 72 hours. N-cadherin and E-cadherin protein expression were determined by western blot analysis. N-cadherin, E-cadherin and GAPDH mRNA expression were determined by real time PCR and relative expression normalized to DMSO samples. The results are representative of 3 independent experiments. (E) OKF6 cells were transfected with 100 nM of siCtrl or 50 nM of siNcad1 and siNcad2 (siNcad), serum starved and treated with TGFβ1 for 24 hours. The cells were added to porous polycarbonate filters coated with type I collagen (5 µg), and allowed to migrate for 24 hours in the presence or absence of TGFβ1. Nonmigratory cells were removed from upper chamber, and migrating cells were counted. *, significantly different from the siCtrl, TGF-b1+ samples with P<0.05. The results are representative of at least three independent experiments.
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