Research Paper Volume 12, Issue 21 pp 21220—21235

Cyclooxygenase-2/sclerostin mediates TGF-β1-induced calcification in vascular smooth muscle cells and rats undergoing renal failure

Figure 5. Effects of TGF-β1 and/or COX-2 on Wnt/β-catenin in VSMCs. (A) Real-time PCR assay results show the effect of TGF-β1 and/or NS398 on mRNA expression of β-catenin in VSMCs cells ("**" p<0.01 vs. control, "#" p<0.05 and "##” p<0.01 vs. TGF-β1 group). (B) Western blot assay results show the effect of TGF-β1 and/or NS398 on β-catenin, sclerostin, p-GSK-3β, and GSK-3β in VSMCs cells; GAPDH was used as a loading control. (C) Quantitative results of Western blot assay show the effect of TGF-β1 and/or NS398 on β-catenin in VSMCs cells ("**" p<0.01 vs. control, "##” p<0.01 vs. TGF-β1 group). (D) Quantitative results of Western blot assay show the effect of TGF-β1 and/or NS398 on sclerostin in VSMCs cells ("**" p<0.01 vs. control, "##” p<0.01 vs. TGF-β1 group). (E) Quantitative results of Western blot assay show the effect of TGF-β1 and/or NS398 on p-GSK-3β and GSK-3β in VSMCs cells ("**" p<0.01 vs. control, "#” p<0.05 and "##” p<0.01 vs. TGF-β1 group). (F) Quantitative results of Western blot assay show the effect of AdTGF-β1 and/or NS398 on TGF-β1 in VSMCs cells ("**" p<0.01 vs. control). (G) IP assay results show the interaction between p-Smad2/3 and p-CREB in VSMCs. (H) ChIP assay results show the recruitment of p-Smad2/3 or p-CREB at the promoter region of sclerostin in VSMCs. (I) ChIP assay results show the recruitment of p-Smad2/3 or p-CREB at the promoter region of β-catenin in VSMCs. NS398: COX-2-specific inhibitor.