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. 2021 Apr 2;11:7470. doi: 10.1038/s41598-021-87091-4

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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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Both AXIN1 and AXIN2 contribute to β-catenin signaling regulation in HCC cell lines. All cell lines were subjected to a β-catenin reporter assay after siRNA-mediated knockdown of AXIN1, AXIN2, a combination thereof or APC. No significant changes in β-catenin reporter activity were observed in the CTNNB1-mutant lines. Among the AXIN1-mutant cells (Hep3B, PLC/PRF/5 and SNU449) both APC and AXIN2 knockdown are equally effective in enhancing signaling. AXIN1 or AXIN2 knockdown in the non-mutant lines (Huh7, HepaRG, SNU182) results in an incomplete increase in reporter activity when compared with APC knockdown, while combined AXIN1/AXIN2 knockdown is similarly effective. For this experiment the WRE/CMV-Renilla ratio for the control siRNA sample-1 was arbitrarily set to 1 for each cell line, after which all β-catenin reporter WRE/CMV-Renilla ratios for the samples were normalized to this control. Only significant differences are shown using an unpaired t-test (mean ± SD, n = 3, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Note the logarithmic Y-axis scale. The result shown here is one representative result, but comparable results were observed in two independent experiments performed on relevant lines (Supplemental Fig. S2).