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. 2020 Dec 10;41(2):898–914. doi: 10.1161/ATVBAHA.120.315506

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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.

This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections.

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Figure 3. — Endoplasmic reticulum (ER) stress modulated vascular smooth muscle cell (VSMC) phenotype. A–C, VSMCs were treated with 2.7 mmol/L Ca²⁺, 2.5 mmol/L PO₄^3-, 0.04 μg/mL tunicamycin (TM), or 0.01 μg/mL thapsigargin (TG), 0.5 mmol/L 4-phenylbutyric acid (PBA) for 8 d in M199 with 5% FBS. Cell death was analyzed with the NucleoCounter NC-3000 using a FLICA caspase assay and propidium iodide staining, where cells are described as healthy, apoptotic, or necrotic. Graph shows mean+SD and individual data points, ANOVA with Tukey post hoc test was performed. Statistical significance is marked for Ca+P vs Ca+P and TM/TG or as indicated with brackets. D, VSMCs were treated with 0.4 μg/mL TM or 0.2 μg/mL TG for 24 h. Osteogenic gene expression in response to ER stress was analyzed by real-time polymerase chain reaction (PCR). Graphs show mean+SD, 1-sample t tests (BMP-2 [bone morphogenetic protein 2], SOX9 [SRY-box transcription factor 9], BSP [bone sialoprotein]), and 1-sample Wilcoxon tests (OSX [Osterix], ALP [alkaline phosphatase]) were performed. E, VSMCs were treated with 2.7 mmol/L Ca²⁺, 2.5 mmol/L PO₄^3-, 0.04 μg/mL TM or 0.01 μg/mL TG, 0.5 mmol/L PBA for 8 d in M199 with 5% FBS. ALP activity was measured using a colorimetric assay. Graph shows mean+SD and individual data points, Kruskal-Wallis test was performed. F, Analysis of SM22α (smooth muscle protein 22α) and SMTN (smoothelin) expression by real-time PCR. One-sample t tests were performed. G, Representative western blots of CNN1 (calponin 1), SM22α, and p-MLC (phosphorylated myosin light chain) and H, quantifications. Graphs show mean+SD and individual data points, 1-sample t tests were performed. CTRL indicates control. Dots denote individual data points, *P<0.05, **P<0.01, ***P<0.001.