Figure 2.

Fibronectin and its receptor, integrin α5β1, specifically increase TGF-β1- and BMP-9-induced Smad1/5/8 phosphorylation. (A) Human microvascular endothelial-1 cells (HMEC-1) were incubated on dishes coated with the indicated concentrations of fibronectin, collagen, and laminin for 15 min and the level of adhesion assessed. The cell adhesion ±s.d. of three independent experiments is presented. (B) HMEC-1 were cultured on Matrigel for indicated times to form tubules. Fibronectin expression levels were assessed, and quantitated with β-actin as a loading control. (C) HMEC-1 were cultured in the dishes coated with 10 μg/ml fibronectin, collagen, or laminin, serum starved for 6 h, treated with 50 pM TGF-β1 for 30 min, the cell lysates analysed with the indicated antibodies. Quantitated data normalized to β-actin from three independent experiments ±s.d. are presented below. (D, E) HMEC-1 were cultured in the dishes coated with indicated doses of fibronectin, serum starved for 6 h, treated with 50 pM TGF-β1 (D) or 2 ng/ml BMP-9 (E) for 30 min, the cell lysates analysed with the indicated antibodies, and the data quantitated below. (F) HMEC-1 were cultured in the dishes coated with 10 μg/ml fibronectin, serum starved for 5 h, pretreated with 10 μg/ml integrin α5β1 function blocking antibody (blk Abs) for 1 h, followed by treatment with 50 pM TGF-β1 or 2 ng/ml BMP-9 for 30 min, and the cell lysates analysed with the indicated antibodies. Figure source data can be found with the Supplementary data.