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. 2020 Apr 24;6:12. doi: 10.1038/s41523-020-0157-z

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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. 1 — a Whole-cell extracts of DCIS.COM and SUM225 cells were immunoprecipitated using an anti-BCL9 antibody, followed by western blot analysis using anti-β-catenin, anti-BCL9, anti-STAT3 and control IgG antibodies. b Densitometry analysis of STAT3 protein in BCL9 pull-down normalized to IgG control in DCIS.COM and SUM225 cells (n = 3, *P < 0.05). c Representative IF images of DCIS.COM MIND xenografts 8 weeks post-intraductal injection. Staining represents P(S727) STAT3 (green), BCL9 (red), and Hoechst (blue). Areas of colocalization of BCL9 and P(S727) STAT3 in the merged image shown in yellow. d PLA using Duolink® PLA kit in 293 T cells transfected with BCL9 overexpression (OE) and either β-catenin, or wild-type (WT)-STAT3, constitutively active (CA)-STAT3, mutant Y705F-STAT3, mutant S727A-STAT3. The antibodies used are indicated in each panel. Anti-β-catenin and anti-BCL9 antibodies were used as PLA-positive controls. Src and β-catenin antibodies were used for PLA-negative control. PLA signals are detected by fluorescence microscopy and appear as red discrete spots. e Bar graphs represent quantification of PLA signals in three biological replicates. PLA signals were analyzed using NIS Elements Analysis Software. f STAT3 reporter assay in BCL9-knockdown (KD) DCIS.COM with and without IL6 stimulation. Data were analyzed using unpaired t-test multi-group comparison (asterisk represents a statistically significant difference; P < 0.05; n = 3 replicates per group). g Whisker plots represent distribution of nuclear BCL9 expression in Patient-derived (PDX) DCIS MIND xenografts that showed invasive progression (n = 7) vs. those that remained non-invasive (n = 4). h, i Pearson correlation between nuclear BCL9 expression and extent of growth (h) or proliferation rate (i) in PDX DCIS MIND xenografts (n = 9). j Whisker plots representing the distribution of nuclear BCL9-P (S727) STAT3 co-expression in progressed (n = 6) vs. non-progressed (n = 4) PDX DCIS MIND xenografts (asterisk represents statistically significant difference in mean values; P < 0.05). k Representative IF images of nuclear BCL9 (red) and P(S727) STAT3 (green) expression and their expression (yellow) in progressed vs. non-progressed primary DCIS MIND xenografts. Data are presented as mean ± SEM.