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. 2021 Sep 10;102(1):80–89. doi: 10.1038/s41374-021-00671-w

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© The Author(s) 2021

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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Fig. 6 — A (a) A scheme of the method to make AM-1 spheroids containing gelatin powders. b Time course analysis of a spheroid formation. Aggregation of cells and gelatin powders are seen within 24 h, and spheroids are formed within 72 h. Scale bars: 500 μm. B Sections of control (a, b) and TGF-β stimulated (c, d) spheroids. Spheroids are showing plexiform formation of AM-1 cells in Hematoxylin-Eosin (H.E.) staining (a, c). Immunohistostaining of Acetylated α-tubulin (Ac-Tubulin) in the control (b) and TGF-β stimulated (d) conditions. The expression of acetylated α-tubulin in AM-1 spheroids is upregulated by the TGF-β stimulation. Scale bars: 100 μm. C (a) Immunofluorescence staining of Ac-Tubulin (red) and E-cadherin (green) in the control and TGF-β stimulated spheroids. Nuclei are stained with DAPI (blue). Scale bars: 100 μm. b Bars indicate relative intensity of Ac-Tubulin in the control and TGF-β stimulated spheroids. The expression of acetylated tubulin is significantly increased in the spheroids especially at the outer layers by the TGF-β stimulation. Statistical significance was set as *p < 0.05 (n = 4).