HS6ST1 silencing reduces TGF-β1 signaling in normal lung fibroblasts. (A) Analysis of phospho- and total Smad2/3 levels at 30 minutes after TGF-β1 stimulation (0.5 ng/ml) by Western blotting. Ratios of P-Smad2/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (with TGF-β1 stimulation), T-Smad2/GAPDH (without TGF-β1 stimulation), and P-Smad3/T-Smad3 (with TGF-β1 stimulation) are shown. White bars, negative control (NC) siRNA–transfected cells; black bars, HS6ST1 siRNA–transfected cells. (B) Analysis of mRNA expression of HS6ST1, collagen I, and α-SMA by quantitative RT-PCR. Fold changes were normalized to the expression of the housekeeping gene 36B4. (C) Analysis of collagen I and α-SMA protein expression at 30 hours after TGF-β1 stimulation (0.5 ng/ml) by Western blotting. (D) Analysis of TβRI, -II, and -III expression at 48 hours after HS6ST1 siRNA transfection by Western blotting. *P < 0.05; **P < 0.01; ***P < 0.001. Data shown are representative of four independent experiments in duplicate.