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. 2018 May 30;8:8434. doi: 10.1038/s41598-018-26501-6

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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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Effect of HMGB1 on normal dermal spheroids. (a) Picrosirius red staining of HMGB1-treated normal dermal spheroids. Collagen deposition increased. The HMGB1 dose dependency of dense and coarse collagen bundles was investigated. (b) Semi-quantitative analysis revealed that treatment with 400 and 1000 ng of HMGB1 significantly increased collagen deposition in normal dermal spheroids by 2.9- and 2-fold, respectively, versus non-treated dermal spheroids (**p < 0.01). (c) Western blotting analysis of HMGB1-treated normal dermal spheroids for the detection of type-I and -III collagen protein expression level (d) Immunohistochemical staining of HMGB1-treated normal spheroid sections for the detection of types I and III collagen, elastin, and fibronectin protein. Increased expression of ECM components (collagen types I and III, elastin, and fibronectin protein) was examined versus nontreated spheroids. Original magnification: 400×. (e to h) Semi-quantitative image analysis of ECM protein expression. The expression levels of type I collagen, type III collagen, elastin, and fibronectin increased significantly versus nontreated spheroids (*p < 0.05; **p < 0.01).