Figure 6

TGF-β promotes tube formation in immortalized pancreatic duct epithelial cell lines. (A) Treatment with 10 ng/mL of TGF-β1 alters the DC-19.18 clone from a sphere- to a tube-forming phenotype. The DC-19.18 clone was cultured in collagen for 8 days in the presence of the following molecules: negative control (mock), 100 ng/mL of Activin-A, 100 ng/mL of Nodal, 100 ng/mL of BMP-2/BMP-7, 100 ng/mL of BMP-4/BMP-7, or 10 ng/mL of TGF-β1. (B) Dose dependency was evident for inducing tube formation of the DC-19.18 clone in the 3D culture condition. (C) An increasing number of tubes was observed with increasing TGF-β1 concentration in the 3D culture of DC-19.18 cells. TGF-β1 was administered to 1000 cells per 20 μL of collagen for 8 days at the following concentrations: mock, 0.1, 1, 10, and 100 ng/mL (n = 3). Multiple comparison (Williams’ method) was used for statistical analysis. **Static ≥ rejection value (2.5%) compared with the controls (mock). Horizontal bars, averages. (D) Treatment with a TGF-β-signaling inhibitor (LY-364947 or SB-431542) alters the tube-forming capacity of the DC-19.12 clone toward the formation of spheres. Increasing concentrations of the inhibitors diminish the tube-forming capacity of the DC-19.12 clone and antagonize treatment with 10 ng/mL of TGF-β1, whereas the sphere-forming capacity of the DC-19.18 clone is not affected by TGF-β-signaling inhibition. (E) The number of spheres and tubes was counted (n = 3). Horizontal bars, means. ^†1Uncountable because of tube-network generation. ^†2Common. (F) Ultrastructural appearance of the tubes and spheres formed by immortalized pancreatic duct epithelial cell lines. The images show the macro appearance of Epon 812-embedded sphere and tube structures (a); the ultrastructural appearance of a sphere structure (b) and a tube structure (c) of DC-19 cells; a tube structure (d) of TGF-β1-treated sphere-forming DC-19.18 cells; and a sphere structure (e) of 10 μM SB-431542-treated tube-forming DC-19.12 cells.