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. 2020 Apr 16;10:6567. doi: 10.1038/s41598-020-63621-4

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© The Author(s) 2020

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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Immunostaining of cell polarity-related proteins. (a) Control spheroids and ECM-loaded spheroids with 0.3 mg/ml Matrigel were fixed two days after injection into MC medium, followed by sectioning and staining for the tight junction-associated proteins ZO-1 (DAB staining) and Claudin-1, the apical marker Ezrin, the bile acid transporter MRP2 (red fluorescence) and staining with Hoechst 33342 for nuclei (blue). Bar = 50 µm. Positive areas for ZO-1, Claudin-1, Ezrin, and MRP2 were quantified and normalized to the nuclear area of Hep G2 (b) and HuH-7 (c) spheroids. The data are shown as the fold of expression in control spheroids. The data are the means ± SEMs, n = 3–4. *p < 0.05. **p < 0.01 by Student t-test.