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 1. Effects of TGF-β1 on calcification in VSMCs and aortic segments. (A) ELISA assay results show the serum levels of TGF-β1 in patients with CKD experienced with dialysis or non-dialysis (n=40) ("**" p<0.01 vs. non-dialysis). (B) Reporter assay results show the effect of high phosphate on TGF-β signaling ("**" p<0.01 vs. control). (C) Real-time PCR assay results show the impact of high phosphate on mRNA expression of TGF-β1 ("*" p<0.05 vs. control, "**" p<0.01 vs. control). (D) Western blot assay results show the effect of high phosphate on TGF-β1; GAPDH was used as a loading control. (E) Quantification results of Western blot assay show the effect of high phosphate on TGF-β1 ("**" p<0.01 vs. control). (F) Real-time PCR assay results show the effect of TGF-β1 recombinant adenovirus on mRNA expression of Runx2, SM22α, and TGF-β1 ("*" p<0.05 vs. control, "**" p<0.01 vs. control). (G) Western blot assay results show the effect of TGF-β1 on Runx2 and α-SMA in VSMCs; GAPDH was used as a loading control. (H) Quantification results of Western blot assay show the effect of TGF-β1 on Runx2 in VSMCs("*" p<0.05 vs. control, "**" p<0.01 vs. control). (I) Quantification results of Western blot assay show the effect of TGF-β1 on α-SMA in VSMCs ("*" p<0.05 vs. control, "**" p<0.01 vs. control). (J) Western blot assay results show the effect of TGF-β1 on OPN in VSMCs; GAPDH was used as a loading control. (K) Quantification results of Western blot assay show the effect of TGF-β1 on OPN in VSMCs ("*" p<0.05 vs. control, "**" p<0.01 vs. control). (L) Quantitative analysis results of Alizarin Red S staining show the effect of exogenous TGF-β1 on inducing mineralization in VSMCs ("*" p<0.05 and "**" p<0.01 vs. control). (M) The Von Kossa staining results show the effect of TGF-β1 on calcification in thoracic segments (Scale=100 μM), Pi: phosphate.