Figure 4. Deficiency of IMD promoted senescence and calcification in VSMCs in vitro. (A) RT-PCR analysis of mRNA level of IMD in VSMCs from WT and IMDSMC-/- mice (n=3). (B) Alizarin red staining (positive staining: red) (scale bar=500 μm) of mice VSMCs (passage 5-6). (C) Western blot analysis of protein levels of α-SMA, SM-22α and RUNX2, and (D–F) quantification (n=3). (G) SA-β-gal staining (blue) (scale bar=500 μm) and (H) quantification of β-galactosidase-positive staining (n=6). WT=wild type. IMDSMC-/-=VSMC-specific IMD-deficient. Con=control. Cal= calcification. Data are mean ± SD. *P<0.05, **P<0.01.