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. 2021 Jan 12;40:30. doi: 10.1186/s13046-020-01800-x

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© The Author(s) 2021

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — SHH002-hu1 inhibits the proliferation of TNBC cell lines and tumor angiogenesis through blocking Wnt/β-catenin pathway. a. The viability of MDA-MB-231/MDA-MB-468 cells was assessed by MTT assay at 48 h after treatment with different concentrations of SHH002-hu1. HEK293T cell and MCF-10A were set as a negative control. SHH002-hu1 specifically inhibited the growth of Fzd7⁺ TNBC cells induced by Wnt3a in a dose-dependent manner. Data were given as the mean ± SD (n = 3), ^**p < 0.01. b. Representative plots showing the apoptosis patterns of SHH002-hu1 treated MDA-MB-231/MDA-MB-468 cells, the percentage of cells in each quadrant was indicated. c. Quantitative analysis of apoptosis assay. Data were presented as the mean ± SD, n = 3, ^**p < 0.01 (MDA-MB-231 cells), vs. the previous group; ^##p < 0.01 (MDA-MB-468 cells), vs. the previous group. d. TOP/FOP ratio in MDA-MB-231/MDA-MB-468 cells (stimulated by Wnt3a) treated with SHH002-hu1 for 24 h. FH535 was set as a positive control. The results from 3 independent experiments are expressed as mean ± SD of fold change, ^*p < 0.05, ^**p < 0.01. SHH002-hu1 effectively inhibited the β-catenin/TCF-4 transcriptional activity induced by Wnt3a. e. SHH002-hu1 repressed the accumulation of β-catenin in the nucleus induced by Wnt3a. IF stainings of β-catenin (green) were shown, and nuclei were counterstained with DAPI (blue), bar = 20 μm. f. SHH002-hu1 blocked nuclear accumulation of β-catenin and phosphorylation of LRP6. MDA-MB-231/MDA-MB-468 cells were treated with Wnt3a and SHH002-hu1, then western blot assay was conducted as indicated. Histone H3 was used as loading control for nuclear proteins, and β-actin was for cytoplasmic proteins. SHH002-hu1 attenuated the Wnt3a induced accumulation of β-catenin and inhibited the induction of phosphorylated (Ser1490) LRP6 by Wnt3a. g, h. HUVECs tube-like photomicrographs and quantitative analysis revealed that the angiogenesis in in vitro model was significantly inhibited by the 8 h-incubation of SHH002-hu1-treated TNBC cells supernatants, bar = 100 μm. The quantitative analysis of HUVECs tube formation (a complete polygon was considered as a tube) was based on the mean of the 5 regions of each group. Data were presented as the mean ± SD, n = 5, ^*p < 0.05. i. Western blot assay indicated that SHH002-hu1 reduced the expression of VEGFA in TNBC cells remarkably