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. 2020 Jun 4;40(7):1763–1776. doi: 10.1161/ATVBAHA.120.314697

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© 2020 The Authors.

Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

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Figure 1. — Increased stiffness promotes in vitro calcification and Runx2 (Runt-related transcription factor 2) nuclear localization in wild-type (WT) vascular smooth muscle cells (VSMCs). A and B, Ddr1^+/+ (WT) or Ddr1^−/− (knockout [KO]) VSMCs cultured in calcifying media were (A) stained with the calcium stain Alizarin Red (n=3) or (B) quantified for deposited calcium by o-cresolphthalein assay (n=3) normalized to total protein quantified by Bradford assay. C, Cytoplasmic (Cyt) and nuclear (Nuc) fractions from WT and KO VSMCs cultured in calcifying media for 2 d were immunoblotted for Runx2 (n=3). D, Nuclear Runx2 and (E) Cyt Runx2 were quantified by densitometry and normalized to Lamin A/C (LMNA) and β-actin respectively. F, WT and KO VSMCs cultured in calcifying media for 2 d were immunostained for Runx2. Scale bar represents 50 μm. G, Runx2 Nuc to Cyt ratio was quantified and expressed relative to 5 kPa WT. *P<0.05, ***P<0.001, bars represent means±SEM. B, D, E, and G, Statistical analysis was performed by 2-way ANOVA with Bonferroni post hoc test. DDR1 indicates discoidin domain receptor-1.