Research Paper Volume 16, Issue 3 pp 2438—2456

Figure 2. Mark4 was upregulated and associated with apoptosis in the aortas of calcified rats and β-GP-cultured VSMCs. (A) Von Kossa staining in the aortas of control rats and calcified rats. (B) Immunohistochemistry of Mark4, Runx2 and cleaved Caspase-3 expression in the aortas of control and calcified rats (n = 3 per group). Scale bars: 20 μm. (C) Alizarin Red S staining of VSMCs cultured with 10 mM β-glycerophosphate (β-GP) medium for 14 days. (D) Quantitative analysis of the calcium content in VSMCs after cultured with β-GP medium for 14 days compared with 0 days. ^**Means p < 0.01. (E) RT–qPCR analysis of Mark4 mRNA expression in VSMCs at 14 days after β-GP induction compared with 0 days. Error bar represent three independent experiments. ^**Means p < 0.01. (F) Western blot and statistical analyses of Mark4, Runx2 and cleaved Caspase-3 expression in VSMCs treated with β-GP for different days. Error bar represents three independent experiments. ^*Means p < 0.05, ^**p < 0.01 vs. control group. (G) Correlation analysis between the protein expression of Mark4 and cleaved Caspase-3. (H) Correlation analysis between the protein expression of Mark4 and osteogenic marker Runx2 in β-GP-induced VSMCs.