Figure 6: MDM2 induces VC.

(a) Adenoviral infection of MDM2 enhanced Pi-induced VC in a dose-dependent manner. Numbers under the horizontal axis are the MOI of adeno-MDM2 (n=3–7 from one to two experimental sets). (b) MDM2 siRNA blunted Pi-induced VC in A10 cells (n=9 from three sets). Either MDM2 siRNA or scramble was transfected with Lipofectamine RNAiMAX. (c) Immunoprecipitation analysis to show that RG 7112 (RG), an MDM2 inhibitor, interfered with the association of HDAC1 with MDM2. Note the physical interaction between HDAC1 and MDM2 (fourth lane) was attenuated by RG treatment (sixth lane). HA-MDM2 and Flag-HDAC1 were transfected and either RG (2.5 μM) or vehicle was treated for 24 h in 293T cells. (d) RG (0.1 μM) blocked the Pi-induced reduction of HDAC1 protein amount in A10 cells. (e) RG attenuated Pi-induced VC in RVSMCs in a dose-dependent manner. Pi-containing media with either RG or vehicle were replaced every 2 days for 6 days and von Kossa staining was performed. Scale bar, 100 μm. (f) Quantification results to show the inhibitory effect of RG on Pi-induced VC. RG (0.5 μM) significantly reduced the calcium deposition in RVSMCs (n=6 from two sets). (g) Dose-dependent attenuation of calcium deposition by RG compound (0.1–3 μM) in human coronary artery smooth muscle cells (HCASMCs). Pi was treated for 3 days (filled circle, n=7 from two sets) or 6 days (open circle, n=3 from one set). *P<0.05, **P<0.01.