Fig. 2

Induction by h-TGF-β1 of KKU-M213 EMT and MMP-9 secretion. KKU-M213 cells were treated with 5 ng/mL h-TGF-β1 with or without 10 µM SB431542 in 0.1% FBS media for the indicated times before assaying for EMT and MMP-9 expression. In EMT assay, cells were lysed and analyzed by immunoblotting using anti-E-cadherin, -phospho-Smad2, -Slug and -β-actin antibodies (a, left panel). The level of vimentin mRNA was determined by SYBR-based qRT-PCR and normalized relative to 18S rRNA (a, right panel). Relative mRNA levels were calculated using 2^−ΔΔCt formula compared to those of controls. After treatment for 24 h, E-cadherin localization was determined by immunofluorescence using anti-E-cadherin antibody (green) and DAPI (blue), then visualized under confocal microscopy (60 × objective lens with 2 × digital zoom), which shows that TGF-β changed E-cadherin localization from membrane to cytoplasm and SB431542 treatment reversed this effect (b). Phase contrast microscopy of the same field is shown in lower panel. MMP-2 and -9 in conditioned media from treated cells were analyzed by gelatin zymography and for uPA by plasminogen-gelatin zymography (c, upper panel). Results were obtained from three independent experiments. *P < 0.05