Recombinant tgf β

In this study, non-stimulated epithelial A549 and Panc-1 cells were used as E-cells. To prepare the mesenchymal-transitioned cancer cells (M-cells), A549 or Panc-1 cells were treated with 5 ng/mL recombinant TGF-β (Pepro Tech, Rocky Hill, NJ, USA) for 48 h then washed with fresh culture medium twice and harvested for subsequent experiments.

Human and murine fibroblasts were isolated and cultured as described previously (Chakraborty et al., 2017 (link); Palumbo-Zerr et al., 2015 (link)). In healthy human dermal fibroblasts, EN1 overexpression was induced by transfection of 0.5 µg of plasmid encoding the human EN1 (Origene). Knockdown of EN1 or of SMAD3 was induced by transfection of 3 µg of EN1-targeting or SMAD3-targeting siRNA using an Amaxa 4D-Nucleofector. Healthy dermal fibroblasts transfected with an equal amount of empty vector or nontargeting siRNA served as controls. In murine fibroblasts isolated from En1^fl/fl mice, Cre-mediated recombination was induced by infection with type 5 adenoviral vectors encoding for Cre recombinase at a multiplicity of infection of 80 (Palumbo-Zerr et al., 2017 (link)). Type 5 adenoviral vectors encoding for LacZ served as controls. In selective experiments, cells were stimulated with recombinant TGFβ (10 ng/ml; PeproTech) for 24 h, unless stated otherwise. In certain experiments, microtubules were stabilized by addition of paclitaxel (1 µM); depolymerized by addition of vinblastine (1 µM); or activity of Rho kinase 1 and 2 was inhibited by Y27632 (1 µM).

A8301 was purchased from Sigma-Aldrich (SML0788; St. Louis, MO, USA), reconstituted in DMSO to a final concentration of 5 mg/mL, and aliquoted and stored at −20°C. Recombinant TGF-β was purchased from PeproTech (100-21B; Rocky Hill, NJ, USA) and stored at −20°C.