A, Deposition of glycosaminoglycan (GAG)-rich matrix detected by Alcian blue stain in micromasses of primary WT and KO VSMCs cultured in DMEM or DMEM-S (DMEM supplemented with ascorbic acid, dexamethasone, and ITS). Graph shows quantitation of extracted Alcian blue dye normalized to total cell number using Crystal violet (N=4). B–C, Wnt16 effect on GAG deposition (B) and expression of chondrogenic markers (C) in KO micromasses cultured in DMEM-S (N=3). Gene expression by real-time PCR normalized to beta-actin is expressed as fold change compared to non-transforming WT VSMCs. D–E, GAG deposition (D) and activation of Smad-dependent (TGFβ/BMP-sensitive) luciferase reporter cells (E) by secreted factors from WT and KO VSMCs in the presence or absence of inhibitors to TGFβ (bold face) or BMP (N=4). *, p<0.05; **, p<0.01.