Figure 8.

Hypoxia-induced TGF-β/Smad signaling activation contributes to VASP up-regulation in HCC. (A) Elisa assay was performed to detect the expression of TGF-β1, 2, and 3 in conditioned media of Hep3B cells exposed to hypoxia. (B) Western blot determined the TGF-β signaling-related protein expression in LO2, Hep3B, and Huh7 cells under normoxia or hypoxia. (C) Western blotting of the TGF-β signaling-related protein expression in LO2, Hep3B, and Huh7 cells following TGF-β treatment. (D) Western blot analysis of VASP expression of Hep3B cells under hypoxia after transfection with Smad3 shRNA or specific p-Smad3 inhibitor SIS3. (E) Upper: ChIP analysis of p-Smad3 binding to VASP promoter in Hep3B cells. Lower: ChIP assay using anti-IgG and anti-p-Smad3 antibodies. (F-G) LO2, Hep3B, and Huh7 cells cultured in normoxic or hypoxic condition were treated with p-Smad3 inhibitor, SIS3, and were subjected to Transwell assay for migration and invasion (F) and Western blot analysis (G) for EMT markers.