Silencing of β4GalT7 antagonized TGF-β1-induced expression of αSMA and TGFβ-1 and activation of Smad signaling pathway.

<p>(<b>A</b>) Lung fibroblasts were transfected with siRNA for β4GalT7 or siRNA control for 48 h and the expression of β4GalT7 was analysed by qPCR. (<b>B</b>) Cells transfected with Siβ4GalT7 or Sicontrol for 48 h were stimulated or not with TGF-β1 (5 ng/ml) for 24 h and the level of PG synthesis was measured by ³⁵Sulfate incorporation during the last 6h of TGF-β1 treatment. The radioactivity associated with GAGs was evaluated by liquid scintillation counting after CPC precipitation and normalized to DNA content. (<b>C</b>) Cells were transfected with Siβ4GalT7 or Sicontrol for 48 h then stimulated or not with TGF-β1 (5 ng/ml) for 3 h and assayed by Western blot to assess the levels of phosphorylation of Smad2 and Smad3. β-actin was used as a loading control. Bar graphs show the expression of phospho-Smad2 and phospho-Smad3 relative to total Smad2/3 level normalized to β-actin and expressed relative to control lysates. Data are expressed as mean ± SD of three separate experiments; *P<0.05 versus control. (<b>D</b>) and (<b>E</b>), the expression of αSMA and of endogenous TGF-β1 were analysed by qPCR after 6 h of TGF-β1 treatment in fibroblasts transfected with Siβ4GalT7 and Sicontrol. The relative expression was normalized with ribosomal protein S29. Data are expressed as mean ± SD of three separate experiments; *P<0.05 versus control.