Figure 2

Cooperative induction of EMT in BEAS-2B cells by TGF-β1 and TWEAK. Confluent monolayers of BEAS-2B cells were cultured for 48 h in the absence (control) or presence of TGF-β1 (10 ng/ml), TNF-α (10 ng/ml), different concentrations of TWEAK (1-100 ng/ml), or TGF-β1 in combination with TNF-α or TWEAK (1-100 ng/ml) as indicated. The levels of E-cadherin (A), ZO-1 (B), N-cadherin (C), and HAS2 (H) mRNA were analyzed by qRT-PCR. Expression levels were normalized to the housekeeping gene GAPDH and calculated as fold induction in comparison to the control. Whole cell lysates were immunoblotted for E-cadherin (D, upper), ZO-1 (E, upper), N-cadherin (F, upper), or vimentin (G, upper). All membranes were re-probed with anti-α-tublin antibody to confirm equal loading. The density of each band was normalized with α-tubulin and quantified by densitometry using NIH ImageJ software (D-G, lower). The capability of cell migration was assessed using a wound-healing assay (I). Data represent the means ± SD of three independent experiments. ^* p < 0.05 compared with the untreated culture. ^† p < 0.05 compared with TGF-β1 alone.