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letter
. 2019 Mar;60(3):367–369. doi: 10.1165/rcmb.2018-0252LE

Figure 1.

Figure 1.

Sustained Smad2 phosphorylation is required for transforming growth factor β (TGF-β)–induced myofibroblast differentiation. Primary cultured human lung fibroblasts were treated with 1 ng/ml TGF-β, 5 μM SB431542, or 10 μM specific inhibitor of Smad3 (SIS3) applied at the times indicated. The cells were then lysed and analyzed for expression or phosphorylation of the desired proteins by Western blotting, or the desired mRNAs by real-time qPCR. (A) Time course of TGF-β–induced Smad2 phosphorylation relative to smooth muscle α-actin (SMA) and collagen 1 (Col1A1) expression. (B) Effect of SB431542 applied 0.5 hour before or 3 hours, 6 hours, and 24 hours after TGF-β treatment on TGF-β–induced Smad2 phosphorylation, and SMA, Col1A1, and fibronectin (FN) expression at 48 hours. (C) Effect of SB431542 applied 0.5 hours before or 3 hours after TGF-β treatment on TGF-β–induced mRNA levels of SMA, Col1A1, and FN at 48 hours. (D) Effect of SIS3 applied 0.5 hours before or 6 hours after TGF-β treatment on TGF-β–induced SMA, Col1A1, and FN expression at 48 hours. (E) Effect of SIS3 applied 0.5 hours before or 6 hours after TGF-β treatment on TGF-β–induced Smad2 phosphorylation at 48 hours or 1 hour of TGF-β treatment as indicated. Data represent the results of at least three independent experiments. *P < 0.01. RLU = relative light units; p-SMAD2 = phospho-SMAD family member 2.