Fig. 1.

β-GP induces calcification and osteogenic differentiation of human VSMCs. Human VSMCs were treated with or without 10 mM β-GP for 12 days (n = 3). a Calcium deposition in cultured VSMCs was detected by alizarin red S staining. Scale bar = 80 mm. b Calcified nodules in cultured VSMCs was detected by alizarin red S staining. Scale bar = 100 µm. c Osteocalcin, Runx2, and α-SMA protein levels were analyzed by Western blot and quantified by densitometry. Statistical significance was assessed using t-test. All graphs show mean + SD. d Calcium content was measured as described in methods. e ALP activity was assessed by spectrophotometry. f Osteocalcin mRNA expression was determined by qRT-PCR. g Runx2 mRNA expression was determined by qRT-PCR. Statistical significance was assessed using t-test or ANOVA. All graphs show mean + SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control. ^##P < 0.01, ^###P < 0.001 vs. β-GP for 3 days