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. 2021 Mar 9;4:312. doi: 10.1038/s42003-021-01825-8

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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 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 PTEN (red) and ATAD1 (blue) transcript levels were measured using qRT-PCR in breast cancer cell lines compared to normal mammary epithelial cells. Error bars are mean ± s.d., triplicate measurements. Significance from normal mammary epithelial cells derived from normal mammoplasty specimen: two-way ANOVA, Dunnett’s correction (P < 0.0001 for all). b Trichostatin A (TSA) and (c) 5-aza-2′-deoxycytidine (AZA) treatment was performed at the indicated doses and transcript levels of PTEN were measured by qRT-PCR in breast cancer cell lines. Error bars: mean ± s.d., triplicate measurements. Significance from Ctrl: one-way ANOVA, Sidak’s correction. d Analysis of co-expression with PTEN including Pearson correlation coefficients and P values in normalized RNA-seq data for previously reported PTEN-repressing genes and genes in the PTEN genomic locus (ATAD1 and KLLN). Cohort includes 818 breast cancer cases³⁴. Significance: two-tailed t test, Pearson correlation (n = 818), black dot indicates a statistically significant result. P values adjusted for multiple comparisons by the Benjamini and Yekutieli method, α = 0.001⁶². e Protein levels of EZH2 and PTEN were measured by immunoblotting in the panel of indicated cell lines. Vinculin was loading control. Quantification is shown below each blot (signal normalized to Vinculin, calculated as a fold change compared to normal breast epithelium isolated from normal mammoplasty specimen). f Immunohistochemical analysis of breast tumors showing PTEN and EZH2 score. Score is expressed as a delta between tumor and normal ducts on the same slide (n = 51 cases). Clinical scoring is used (0–3+ range based on intensity). Error bars: mean ± s.e.m. g Reperentative photos of IHC staining in normal ducts (BRP ID# 6621), non-TNBC (BRP ID# 6617), and TNBC (BRP ID# 6706), showing PTEN, EZH2 and H&E. ×5 magnification, Scale bar: 500 µm. h Boxplots of EZH2 RNA-seq z-scores in the indicated subtypes of breast cancer³⁴, number of cases indicated for each subtype (total n = 969). Significance from ER+: one-way ANOVA, Dunnett’s correction. (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05).