Fig 4. Alprostadil inhibits TGF–β1–induced changes in NHLF by activating both, the EP₂ and the EP₄ receptor,without affecting baseline α–SMA and COL1 levels in non–stimulated NHLF.

NHLF fibroblasts were incubated with dilution series of alprostadil (0.01–10,000 nM) at t = 0 h and then stimulated (A–C) or not (D–F) with TGF–β1 (5 ng / ml). Non–stimulated NHLF cells (0 ng / ml TGF–β1, 0 nM alprostadil, baseline) and 5 ng / ml TGF–β1–treated (0 nM alprostadil; vehicle) are depicted in green and red, respectively (A, D). Concentration response curves of alprostadil in presence (B) and absence (E) of 5 ng / ml TGF–β1 where then generated with baseline subtracted impedance values at t = 20 h post TGF–β1 addition. (G) At t = 0 h NHLF fibroblasts were incubated with 10,000 nM of the EP₂ prostaglandin receptor antagonist, the EP₄ receptor antagonist, and of both the EP₂ and the EP₄ receptor antagonists in the absence or in presence of 10 nM alprostadil and 5 ng / ml TGF–β1. At t = 48 h after TGF–β1 (C, G) or vehicle (F) addition the cells were lysed and α–SMA and COL1 were quantified by MS / MS. Bars represent protein data normalized to tubulin. Number of sample (n = 1) is shown in each figure A, D, E, F. Bars represent mean of n = 2 samples in B, C and G.