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. 2021 Sep 13;11:18172. doi: 10.1038/s41598-021-97457-3

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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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Cell cycle analysis. Cancer cells were treated with various concentrations of biobutyrate for 24, 48, and 72 h. The treatment of PBS was used as a control. (A) Flow cytometric analysis of biobutyrate-treated cells. After the treatment of biobutyrate, cells were analyzed by FACSCanto flow cytometer. (B) Quantification of cells at different stages of cell cycle. Data from three independent experiments were analyzed by the GraphPad Prism 7 software (*p < 0.05, **p < 0.01 and ***p < 0.001 vs. control based on one-way ANOVA) and graphs were prepared using SigmaPlot 10.0 (Systat Software, San Jose, CA). (C) Immunoblotting analysis of the cell cycle regulatory proteins. HT29 cells were treated with various doses of biobutyrate for 24 h. (D) Quantification of the cell cycle regulatory proteins. Full-length blots are presented in Supplementary Fig. 3C. Data from three independent experiments were analyzed by the GraphPad Prism 7 software (*p < 0.05, **p < 0.01 and ***p < 0.001 vs. control based on one-way ANOVA) and graphs were prepared using SigmaPlot 10.0 (Systat Software, San Jose, CA).