Figure 5. IMD1-53 inhibited aging-associated vascular calcification by increasing sirt1 expression and deacetylase activity. (A) Western blot analysis of protein levels of sirt1, acetylation p53 (acetyl-p53), and total p53 (p53) in rat aortas, and (B, C) quantification (n=3). (D) Immunofluorescence staining for α-SMA (red) and sirt1 (green) in rat aortas. Nuclei were stained with Hoechst 33342 (blue). Merged images (α-SMA, sirt1 and nuclei) are shown (Scale bar=200 μm). (E) Western blot analysis of protein levels of sirt1, acetyl-p53, and p53 in rat VSMCs and (F, G) quantification (n=3). (H) Western blot analysis and quantification of protein level of sirt1 in WT and IMDSMC-/- mouse VSMCs (n=3). (I) Alizarin red staining (red) (Scale bar=500 μm) and (J) SA-β-gal staining (blue) (Scale bar=100 μm) of calcified-rat senescent VSMCs treated with IMD1-53 plus sirt1 siRNA or negative siRNA, and (K) quantification of β-galactosidase-positive staining (n=6). (L) Western blot analysis of protein levels of sirt1, acetyl-p53, p53, p16, RUNX2 and MGP and (M–Q) quantification (n=3). For in vivo experiments, Y=young rats; O=old rats; YV=young+VDN; OV=old+VDN; OVI=old+VDN+IMD1-53. For in vitro experiments, Y=young VSMCs; S=senescent VSMCs; YC=young+calcification; SC=senescence+calcification; SCI=senescence+calcification+IMD1-53. WT=wild type. IMDSMC-/-=VSMC-specific IMD-deficient. Con=control. Cal= calcification. neg si=negative siRNA. sirt1 si=sirt1 siRNA. Cal=calcification. Data are mean ± SD. *P<0.05, **P<0.01.