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. 2021 Jan 12;11:515. doi: 10.1038/s41598-020-80019-4

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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The size of spheroids sharply increases and correlates with the expression of proliferation genes. (A) TPEF images of 3D cultured PHH at d2, d6 and d10 of culture (10× magnification, z projection). Scale bar = 100 μm. (B) The viability was assessed by measuring the cellular ATP content in 3D cultured PHH at different times of culture (n = 3) (Mean ± SD, **p < 0.01, ***p < 0.001, one-way ANOVA test). (C) Phase-contrast imaging and HES staining of spheroids at d2, d4, d6 and d10 of culture. Scale bar = 50 μm. Quantification of the mean diameter size of spheroids during the time of culture. Diameters of spheroids are from three independents experiments (Mean ± SD). (D) Boxplots extracted from transcriptomic analysis of the expression of the main genes involved in the proliferation of epithelial cells (AURKB, CCND1, CDK1, MIK67, PKL1, PTTG1, CENPF). (*p < 0.05, Mann–Whitney U test).