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. 2018 Jan 12;8:658. doi: 10.1038/s41598-017-19092-1

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© The Author(s) 2018

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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(A) Typical images of calcification of HASMCs induced by IL-24 and inhibition of calcification by anti-IL-24 antibody. To confirm the effect of IL-24 on HASMC calcification, the cells were incubated with the calcification medium for 15–21 days in the presence of recombinant human IL-24 (0 or 5 ng/mL) and TNF-alpha stimulation (0 or 1 ng/mL) with or without anti-IL24 antibody (0.5 μg/ml). Mineralized nodules of cells were stained with Alizarin red, and typical calcification images in HASMCs are shown. The anti-IL-24 antibody inhibited the calcification induced by IL-24 stimulation. Black bars indicate 500 micrometers. (B) Quantification of calcification of HASMCs induced by recombinant IL-24 or TNF-alpha and inhibition of the calcification by anti-IL-24 antibody by ImageJ software. The calcification areas of HASMCs stained with Alizarin red were quantified by ImageJ software. Recombinant IL-24 significantly induced HASMC calcification (P < 0.05), and the anti-IL-24 antibody significantly inhibited the calcification (P < 0.01). The calcification induced by both recombinant IL-24 and TNF-alpha was also significantly inhibited by the anti-IL-24 antibody (P < 0.05). These experiments used two cell lines of HASMCs.