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 3. Effects of high phosphate and/or TGF-β1 on Wnt/β-catenin in VSMCs. (A) Luciferase reporter assay results show the effect of high phosphate on Wnt/β-catenin signal ("*" p<0.05 and "**" p<0.01 vs. control). (B) Western blot assay results show the effect of high phosphate on β-catenin, p-GSK-3β, and GSK-3β in VSMCs cells; GAPDH was used as a loading control. (C) Quantification results of Western blot assay show the effect of high phosphate on β-catenin in VSMCs cells ("**" p<0.01 vs. control). (D) Quantification results of Western blot assay show the effect of high phosphate on p-GSK-3β and GSK-3β in VSMCs cells ("*" p<0.05 and "**" p<0.01 vs. control). (E) Real-time PCR assay results show the effect of TGF-β1 on mRNA expression of β-catenin in VSMCs cells ("**" p<0.01 vs. control). (F) Immunofluorescent staining results show the effect of TGF-β1 on β-catenin and its localization in VSMCs (Scale=50 μM). (G) Western blot assay results show the effect of TGF-β1 on sclerostin, β-catenin, and c-Myc in VSMCs cells, GAPDH was used as a loading control. (H) Quantitative results of Western blot assay show the effect of TGF-β1 on β-catenin in VSMCs cells ("*" p<0.05 and "**" p<0.01 vs. control). (I) Quantitative results of Western blot assay show the effect of TGF-β1 on sclerostin in VSMCs cells ("*" p<0.05 and "**" p<0.01 vs. control). (J) Quantitative results of Western blot assay show the effect of TGF-β1 on c-Myc in VSMCs cells ("*" p<0.05 and "**" p<0.01 vs. control). (K) Real-time PCR assay results show the effect of high phosphate and/or LY364947 on mRNA expression of β-catenin in VSMCs cells ("**" p<0.01 vs. control; "#" p<0.05 and "##” p<0.01 vs. high phosphate group). (L) Western blot assay results show the effect of high phosphate and/or LY364947 on β-catenin, p-GSK-3β, and GSK-3β in VSMCs cells; GAPDH was used as a loading control. (M) Quantitative results of Western blot assay show the effect of high phosphate and/or LY364947 on β-catenin in VSMCs cells ("**" p<0.01 vs. control; "#" p<0.05 and "##” p<0.01 vs. high phosphate group). (N) Quantitative results of Western blot assay show the effect of high phosphate and/or LY364947 on p-GSK-3β and GSK-3β in VSMCs cells ("**" p<0.01 vs. control; "##” p<0.01 vs. high phosphate group). (O) Quantitative analysis results of Alizarin Red S staining show the effect of TGF-β1 and/or sclerostin on mineralization in VSMCs ("**" p<0.01 vs. control; "#" p<0.05 vs. TGF-β1 group). Pi: phosphate, LY364947: TGFβRI-specific inhibitor.