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. 2020 Jun 30;5:105. doi: 10.1038/s41392-020-00218-3

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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 — FOXM1D directly interacts with p110 and p85 subunits of PI3K and regulates PI3K activity. a The co-IP and silver staining assays to identify the FOXM1D binding proteins in the lysates from HeLa cells expressing FLAG-FOXM1D. b, c The co-IP (b) and ICC (c) assays to determine the interaction between FOXM1D and p110α/β/δ. Scale bar, 25 μm (c). d, e Identification of the mutual binding sites of FOXM1D and p110β. Co-IP assays were performed of the lysates from 293FT cells expressing FLAG-FOXM1D or FLAG-tagged truncated mutants of FOXM1D (d) using a FLAG antibody (e). The obtained samples were detected by IB using FLAG and p110β antibodies (e). f–h The co-IP (f, g) and ICC (h) assays to determine the interaction between FOXM1D and p85α. Scale bar, 10 μm (h). i Identification of the mutual binding sites of FOXM1D and p85α. Co-IP assays were performed of extracts from the 293FT cells expressing FLAG-FOXM1D or FLAG-tagged truncated mutants of FOXM1D (d) and HA-p85α-expressing plasmids using a FLAG antibody (i). The obtained samples were detected by IB using FLAG and HA antibodies (i). j, k The immunoblotting assays to determine the activation of PI3K/AKT/GSK3β/Snail signaling axis in SW480 cells with ectopic FOXM1D expression (j) and in LoVo cells with knocked-down FOXM1D expression (k). Error bars: mean ± SD (n = 3); Student’s t-test; *P < 0.05, **P < 0.01; ***P < 0.001. l The working model. FOXM1D binds to p85 in its junction region between iSH2 and cSH2 via its exon II- and III-encoding region, while binds to p110 kinase domain via its exon VIII-encoding region; therefore, the intermediary FOXM1D generates an effect of steric hindrance between p110 and p85, thus relieving the inhibitory effect of p85 on p110 kinase activity and enhancing the PI3K/AKT activation upon upstream signaling stimulation, which leads to increased phosphorylated GSK3β, stabilized Snail and accumulated nuclear Snail, contributing to the transcriptional repression of E-cadherin and elevation of vimentin, and cancer metastasis. GF growth factor, RTK receptor tyrosine kinase