Figure 6.

RhoA (ras homolog family member A) and actomyosin contractility promote Runx2 (Runt-related transcription factor 2) nuclear (Nuc) localization and expression as well as vascular smooth muscle cells (VSMC) calcification. A, Wild-type (WT) VSMCs cultured in calcifying media for 2 days with ACT (Rho activator II) or C3 (C3 exoenzyme) were stained with AlexaFluor488 phalloidin and Runx2 primary antibody with AlexaFluor568 anti-rabbit secondary antibody and imaged at ×200 magnification by confocal microscopy (n=3). Scale bar represents 50 μm. B, Nuc to cytoplasmic (Cyt) ratio was calculated with 10 to 30 cells per treatment group for each replicate. Ratios are expressed relative to control (Ctrl). C, WT VSMCs cultured in calcifying media for 2 days with ACT or C3 were immunoblotted (n=3). D, Runx2 expression was quantified by densitometry and normalized to β-actin. E, WT and knockout (KO) VSMCs were cultured in calcifying media for 12 days with or without blebbistatin before staining with Alizarin Red (n=3). F, Higher magnification images were taken from the wells shown in E. Scale bar represents 500 μm. B and D, *P<0.05, **P<0.01, bars represent means±SEM. Statistical analysis was performed by 1-way ANOVA with Bonferroni post hoc test. DDR1 indicates discoidin domain receptor-1.