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. 2018 Feb 21;8:3377. doi: 10.1038/s41598-017-17540-6

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Lysosomal function is increased in calcifying VSMCs. Confluent rat aortic VSMCs were cultured on a chamber slide in a Pi + Ca²⁺ (3.5 mM phosphate/3 mM calcium) calcification medium for 7 days, the function of lysosome was evaluated by the LysoTracker red and cathepsin B magic red staining. (A and C) Representative immunofluorescent staining images and quantitative results showing increased LysoTracker intensity and decreased SM22α expression in calcifying VSMCs. (C–D) Immunofluorescent staining images and quantitative results showing effects of calcification medium on cathepsin B activity and SM22α expression. The fluorescent intensity of lysosomes was analyzed using Image J software. At least 5 fields per condition and 3 independent experiments were performed. All Values are presented as mean ± SD. *P < 0.05.