Figure 3

1,25(OH)₂D₃ inhibits phosphorylation of smad2/3 in nasal polyp-derived fibroblasts. Nasal polyp-derived fibroblasts were treated with TGF-β1 and/or 1,25(OH)₂D₃ up to 4 hours. (A) Expression levels of phosphorylated Smad2/3 were determined by Western blotting. (B) Specific VDR siRNA (10 nM) was transfected in NPDFs and the inhibitory effect of VDR siRNA was confirmed by Western blotting. (C) Transfected NPDFs with VDR siRNA were treated with or without 1,25(OH)₂D₃ (100 nM) for 1 hour and cells were stimulated with TGF-β1 (5 ng/ml) for 4 hours and 72 hours. Phosphorylated Smad2/3 and α-SMA, fibronectin was determined by Western blotting. (D) NPDFs were treated with TGF-β1 (5 ng/ml) and/or 1,25(OH)₂D₃ (100 nM) or SIS3 (a Smad3-specific inhibitor, 3 μM). Expression levels of phosphorylation of Smad2/3 (4 hours) and α-SMA and fibronectin (72 hours) were determined by Western blotting. Expression levels of the housekeeping GAPDH protein were utilized as internal controls. A representative experiment and quantitative determination of protein levels are shown. (E) The amount of total soluble collagen in culture media was quantified by the Sircol collagen assay. (F) Representative fluorescein immunocytochemistry for fibronectin (green) with nuclear DAPI (blue). Scale bar = 50 μm. All data are presented as mean ± SEM. Four primary cell lines from different donors were used. All experiments were performed in at least triplicate and repeated at least three times. *p < 0.05 vs. control, ^†p < 0.05, ^††p < 0.01 vs. TGF-β1.