Fig. 6

Noggin, BMP2 antagonist, suppresses VSMC calcification by inhibiting Smad1/5/8 signaling. VSMCs pretreated with Noggin (100 ng/ml) or vehicle for 2 h followed by incubation with 10 mM β-GP. a Alizarin Red staining (×100). b Quantification of Alizarin Red staining. c Calcium contents measured and normalized by protein content of cell lysates. mRNA expression of (d) BMP2, (e) Msx2, (f) Runx2, and (g) osteocalcin analyzed by rt-PCR. h Protein expression of BMP2 and phospho-Smad1/5/8 performed by western blot analysis. β-GP beta-glycerophosphate, DMEM Dulbecco’s Modified Eagle’s Medium, DMEM^β-GP DMEM medium containing 10 mM β-GP, DMEM^Noggin+β-GP DMEM medium containing 10 mM β-GP and 100 ng/ml Noggin, Msx2 msh homeobox 2, NC DMEM medium, OD optical density, Runx2 Runt-related transcription factor 2