Fig. 5

Impact of exogenous FGF1 + heparin on the regulation of pro-fibrotic proteins. Western blots were performed on lysates harvested from IPF and non-IPF (donor) fibroblasts (2 technical replicates of 6 independent biological samples). Cells were starved for 24 h (lane 1) and then treated once daily for two days with heparin (25 ng/mL) (lane 2) recombinant human FGF1 (25 ng/mL) (lane 3), FGF1 + heparin together (lane 4), the FGFR inhibitor, PD173074 (0.1 μM resuspended in DMSO) (lane 5), DMSO (0.1 μM) only (lane 6), or FGF1 + heparin + PD173074 (lane 7) simultaneously. Collagen 1a1 (COL1a1, bands present at both 170 and 140kDA) was blotted against b-tubulin (TUBB1) (a) and smooth muscle actin (ACTA2/α-SMA) was blotted against GAPDH (b). Densitometry plots of arbitrary units indicated that in donor fibroblasts, COL1a1 was not significantly regulated (b,c) and neither was ACTA2 (α-SMA) (a,d). Untreated lysates of IPF fibroblasts displayed more collagen than in donor controls (a). COL1a1, especially the 170kDA band, was strongly reduced in FGF1 + heparin groups (b,c) while ACTA2 was not regulated (a,d). Next p-ERK1/2 was blotted over total ERK (e,f,g), and Fascin (e,h), a cell invasion/migration marker. In donor fibroblasts, p-ERK1 signal was significantly increased in the FGF1 alone group and reduced in the presence of the inhibitor (e,f). The p-ERK2 signal was increased in donor fibroblasts where exogenous FGF1 or heparin or both were added and p-ERK2 was attenuated in the presence of the inhibitor (e,g). In IPF fibroblasts, p-ERK1 was significantly activated when exposed to FGF1 alone or FGF1 + heparin and attenuated when the inhibitor was present (e,f) and p-ERK2 was similarly regulated (e,g). A trend towards an increase in the cell migration marker Fascin was observed in FGF1 + heparin treated donor and IPF fibroblasts (e, h)