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. 2020 Sep 10;8:862. doi: 10.3389/fcell.2020.00862

FIGURE 5.

FIGURE 5

Fetuin A attenuates calcification in HAVICs and is associated with inactivation of Wnt signaling genes. (A) Representative images of endogenous Fetuin A in HAVICs localized to the golgi apparatus (G), endoplasmic reticulum (ER), and vesicles (V). Arrows point to regions of interest. Scale bar: 20 μm. (B) Representative Alizarin Red S staining images of HAVICs treated with different concentrations of Fetuin A. (C) Quantification of calcium deposition using acid extraction following Alizarin Red S stain of HAVIC relative to Crystal Violet stain. The data are represented as mean ± SEM. Significance level was set at P < 0.05. *P < 0.05, **P < 0.01 compared to the specified group; n = 6 HAVIC lines were used for each group. One-way ANOVA followed by Tukey’s post hoc multiple comparisons test was used for statistical analysis. (D) mRNA expression of osteogenic and Wnt signaling genes after treatment with Fetuin A. The data are represented as mean ± SEM. Significance level was set at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control groups; n = 6 HAVIC lines were used for each group. Students t-test was used for statistical analysis. (E) Representative Western blot of phosphorylated β-catenin and β-catenin protein expression in HAVICs treated with 10 μM Fetuin A in OSM. (F) Densitometry analysis of phosphorylated β-catenin and β-catenin protein expression in HAVICs treated with 10 μM Fetuin A in OSM. The data are represented as mean ± SEM. Significance level was set at P < 0.05. **P < 0.01, ***P < 0.001; n = 2 for each group. Students t-test was used for statistical analysis. (G) mRNA expression of intracellular Wnt signaling genes after treatment with Fetuin A. The data are represented as mean ± SEM. Significance level was set at P < 0.05. ***P < 0.001, ****P < 0.0001 compared to control groups; n = 3 HAVIC lines were used for each group. One-way ANOVA followed by Tukey’s post hoc multiple comparisons test was used for statistical analysis. AVS, aortic valve stenosis; HAVICs, human aortic valve interstitial cells; OSM, osteogenic media; FA, Fetuin A.