Research Paper Volume 12, Issue 7 pp 5651—5674

Intermedin1-53 attenuates aging-associated vascular calcification in rats by upregulating sirtuin 1

Figure 3. IMD1-53 inhibited senescence-associated calcification in rat VSMCs. (A) Alizarin red staining for rat VSMCs (positive staining: red) (Scale bar=500 μm). (B) SA-β-gal staining (blue) (Scale bar=100 μm) and (C) quantification of β-galactosidase-positive staining in rat VSMCs (n=6). (D) Calcium content assay (n=6) and (E) ALP activity assay (n=6) of rat VSMCs. (F) Western blot analysis of protein levels of smooth muscle 22 alpha (SM-22α), alpha smooth muscle actin (α-SMA), bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), matrix γ-carboxyglutamic acid (Gla) protein (MGP), and cyclin-dependent kinase inhibitors p16 and p21 in rat VSMCs, and (GM) quantification (n=3). Y=young VSMCs; S=senescent VSMCs; YC=young+calcification; SC=senescence+calcification; SCI=senescence+calcification+IMD1-53. Data are mean ± SD. *P<0.05, **P<0.01.