FIGURE 2.

Fibronectin induces AKT activation downstream from FGFR1. A, serum-starved TSECs were plated on FN-coated or PBS-coated dishes overnight and then stimulated with 1 ng/ml FGF2 or vehicle for 15 min. Phosphorylation of Tyr-653/654-FGFR1, AKT, and ERK and total FGFR1, AKT, and ERK were evaluated by Western blot analysis from protein lysates. Top, representative blots; bottom, quantification of phosphorylation of AKT and ERK from multiple blots, normalized by the ratio in the non-stimulated cells on a PBS-coated dish (n = 3; *, p < 0.05 between depicted groups). B, TSECs were transfected with retrovirus encoding FGFR1-WT or YFP as control, with aliquots of FGFR1-WT-transfected cells also transfected with lentiviral FGFR1 shRNA. 3 days after transfection, cells were serum-starved and plated on FN-coated or PBS-coated dishes. Phosphorylation of FGFR1 at Tyr-653/654 and AKT and total FGFR1 and AKT were evaluated. Left, representative blots; right, quantification from multiple blots, normalized by the ratio in the YFP-retrovirus-infected cells on an FN-coated dish (n = 3; *, p < 0.05). C, endogenous FGFR1 in TSECs was silenced by FGFR1 siRNA transfection (20 nm, 3 days, with non-targeting siRNA as control), and cells were replated on an FN- or PBS-coated dish overnight in basal medium. Phosphorylation of FGFR1 and AKT, total FGFR1 and AKT, and GAPDH were evaluated by Western blot. Top, representative blots; bottom, quantification from multiple blots, normalized by the ratio in the non-targeting siRNA-transfected cells on a PBS-coated dish (n = 3; *, p < 0.05). D, TSECs were serum-starved and seeded on FN- or PBS-coated dishes in basal medium overnight and then treated with PD173074 (40 nm) or DMSO as control for 2 h. Phosphorylation of FGFR1 and AKT and total FGFR1 and AKT were evaluated by Western blot. Left, representative blots; right, quantification from multiple blots, normalized by the ratio in the DMSO-treated cells on a PBS-coated dish (n = 4; *, p < 0.05). Error bars, S.E.