Fig. 2. Assessment of effects of TGF-β1 in MTEC on expression of mesenchymal and epithelial markers. (A) Evaluation of collagen type 1a1, fibronectin and E-cadherin via immunofluorescence. MTEC were exposed to 5 ng/ml TGF-β1 every other day for 14 days, and prepared for immunofluorescence. Magnification 400x. DAPI illustrates nuclei. (B) MTEC were exposed to 5 ng/ml TGF-β1, and expression of PAI-1 was assessed in cell culture media 24 hours later. -SMA protein expression was determined in cell lysates following exposure to 0.5 or 5 ng/ml of TGF-β1 for 6 days. Smad3 represents loading control. Different lanes shown represent results from independent samples. (C) Evaluation of mRNA levels of mesenchymal or epithelial genes in MTEC exposed to TGF-β1 treatment (5 ng/ml, every other day for 10 days). To evaluate reversibility of TGF-β1 responses, MTEC were maintained in culture for 6 additional days in the absence of TGF-β1. mRNA expression was evaluated by real-time PCR analysis, and data normalized to the housekeeping gene HPRT. Results are expressed as fold changes from controls. ^* P<0.05, ANOVA versus control-treated, ^** P<0.05, ANOVA versus TGF-β1-treated.