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. 2020 Aug 24;11(8):703. doi: 10.1038/s41419-020-02838-7

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© The Author(s) 2020

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. 4 — a MCF-7 and MDA-MB-231 cells were treated with varying concentrations of MLN4924 (0, 0.1, 0.5, 1.0 μM) for 48 h and analyse for the cell proliferation rates with MTT assay. Graphical representation was the optical density (wavelength = 570 nM) of the MLN4924-treated MCF-7 and MDA-MB-231 cells relative to the untreated control counterparts. (N = 3). b MCF-7 and MDA-MB-231 cells were treated with varying concentrations of MLN4924 (0, 0.1, 0.3 and 1.0 µM) for 10 days. The representative images showed cell clonogenic survival of the MLN4924-treated MCF7 and MDA-MB-231 cells (left panels). Data represent means ± SD. (n = 3; t tests). Significant *P < 0.05; **P < 0.01; ***P < 0.001. c MDA-MB-231 cells were treated with varying concentrations (0, 0.1, 0.5 and 1.0 µM) of MLN4924 and injected onto the chorioallantoic membrane of 9 days old fertilised chick egg xenograft model (left panel). The representative images showed the dissected tumour after a 5 days incubation (central panel). (N = 3). Data are representative of three independent experiments. Data represent means ± SD. (n = 3; t tests). Significant **P < 0.01; ***P < 0.001. d MDA-MB-231 cells were treated with 1 µM MLN4924 for 24 h and the protein expression of p21^Waf1/Cip1 and p27^Kip1 were analysed by western blotting. α-Tubulin was used as a loading control. (N = 3). e MDA-MB-231 cells were transfected with ERRβ shRNA and/or treated with 1.0 µM MLN4924. The representative images showed cell clonogenic survival of the MLN4924-treated, with and without ERRβ-knockdown, MDA-MB-231 cells. (N = 3) (top right). Data represent means ± SD. (n = 3; t tests). Significant; *P < 0.05; ns non-significant. (bottom right). f MDA-MB-231 cells were treated with 1.0 µM MLN4924 in the presence or absence of p21^Waf1/Cip1 shRNA-mediated knockdown. The protein expression of p21^Waf1/Cip1 was analysed by western blotting with or without p21^Waf1/Cip1 depletion in the presence of 1.0 µM MLN4924. GAPDH was used as a loading control. The representative images showed cell clonogenic survival of the MLN4924-treated, with and without p21^Waf1/Cip1-knockdown, MDA-MB-231 cells. (N = 3) (middle). Data represent means ± SD. (n = 3; t tests). Significant; *P < 0.05 (right).