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. 2019 Mar 20;9:4905. doi: 10.1038/s41598-019-41355-2

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© The Author(s) 2019

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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Immunofluorescence staining and colocalization analysis of MMP2 and TIMP3, and of SH with MMP2 and TIMP3. 7,000 hBMSC/cm² were plated in basic medium and treated with CS, HA, SH, and HE (200 µg/mL each) (A) or with ATTO655-SH (violet) (B). At day 8 after plating cells were fixed with paraformaldehyde and stained for MMP2 (green), TIMP3 (red), and nuclei (blue); scale bars 50 µm (A,B). (B) Images in line from left to right show merged MMP2/TIMP3 (yellow), SH (violet), merged SH/MMP2 (white), merged SH/TIMP3 (purple), and merged SH/MMP2/TIMP3 (pale pink). The extent of colocalization of MMP2 with TIMP3 (C) (according to images in A) and of SH with either MMP2 (D) or TIMP3 (E) (according to images in B) was calculated as described. Pearson correlation coefficient (summarized signal) values >0.5 (dotted line) indicate a high probability that pixels of both channels are overlay. Manders’ coefficients M1 and M2 (M1 indicates the overlap of SH signal (violet channel) with MMP2 signal (green channel) (D,E). Values are given as mean ± SEM; significant differences of treatment vs. Ctrl were analyzed by one-way ANOVA/Bonferroni’s post-test and are indicated with ***(p < 0.001), n = 8.