Figure 6. Involvement of signaling pathways in IMD1-53 upregulating sirt1 in senescence-associated VSMC calcification. (A) Western blot analysis of IMD17-47 (10-6 mol/L) blocking the effect of IMD1–53 on sirt1 protein level in calcified-rat senescent VSMCs, and (B) quantification (n=3). (C) Western blot analysis of protein levels of sirt1, phosphorylated-AMP-activated protein kinase (p-AMPK) (Thr172), AMPK, phosphorylated-protein kinase A (p-PKA) (Thr197), PKA, phosphorylated-protein kinase B (p-Akt) (Ser473), Akt in rat senescent VSMCs preincubation with or without phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, AMPK inhibitor Compound C or PKA inhibitor H89 (all 10 μmol/L) before IMD1–53 administration and calcification induction, and (D–G) quantification of (D) sirt1 (n=3), (E) p-AMPK/AMPK (n=3), (F) p-PKA/PKA (n=3), (G) p-Akt/Akt (n=3). (H) Alizarin red staining (Scale bar=200 μm), and (I) quantification of calcium deposition-positive staining (red) (n=6). (J) SA-β-gal staining (Scale bar=100 μm), and (K) quantification of β-galactosidase-positive staining (blue) (n=6). Con=control. Cal=calcification. Data are mean ± SD. *P<0.05, **P<0.01.