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. 2020 Oct 5;11:4977. doi: 10.1038/s41467-020-18701-4

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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. 7 — a Fold change in sphere numbers of pre-malignant (MCF 10A) and tumorigenic cell lines (MCF-7, MDA-MB-231) without (MCF 10A parental, n = 8; MDA-MB-231, n = 6) or with mutational activation of PIK3CA (MCF 10A PIK3CAE545K/+, n = 7; MCF-7, n = 6) cultured in the presence or absence of HIL6. Note that MCF 10A PIK3CAE545K/+ cells are isogenic to MCF 10A parental; n.s. = non-significant. b Western blot analyses showing phosphorylation of STAT3Tyr705, AKTSer475 and ERK1/2Thr202/Tyr204 in MCF 10A or MCF 10A PIK3CAE545K/+ cells cultured without or with HIL6 for the indicated time. For quantification, the signal of the phosphorylated protein and total protein was normalized to α-tubulin, then the ratio of phosphorylated to total protein was calculated. The graphs show the fold change in signal ratio over time relative to the control (unstimulated MCF 10A wt = 1). c Sphere numbers of the isogenic cells MCF 10A parental (n = 8) and MCF 10A PIK3CAE545K/+ (n = 7) cultured in the absence of HIL6. d Cytokeratin 8/18/19+ DCCs from BM of non-metastasized (M0-stage) HR-positive breast cancer patients and CD45-/EpCAM+/cytokeratin 8/18/19+ CTCs isolated from peripheral blood of metastasized (M1-stage) HR-positive breast cancer patients were sequenced for hotspot-mutations in PIK3CA (Exon 9: E545K, E542K; Exon 20: H1047R, H1047L, M1043I). P values in a, c two-sided Student’s test; d two-sided Fisher’s exact test. All error bars correspond to standard deviation (Mean ± SD).