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. 2019 Jun 28;10:2860. doi: 10.1038/s41467-019-10743-7

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© The Author(s) 2019

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. 3 — Death effector domain-containing DNA-binding protein (DEDD) mediates heat-shock cognate 71 kDa protein (HSC70)-dependent G1/S progression and cyclin D1. a, Left: immunohistochemistry (IHC) staining of DEDD in triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) tissue. Right: Schematic of 3×-FLAG-tagged wild-type DEDD nuclear localization sequence (NLS) location and its mutated form. b, Left: Western blot showing FLAG-DEDD expression in 293FT cells with or without cytosol shuttle inhibitor leptomycin treatment (10 μg/mL, 3 h). Right: Representative flow cytometry biaxial plots of 293FT cells showing 5-ethynyl-2′-deoxyuridine (EdU) incorporation with or without expression of wild-type DEDD or cytosolic DEDD (∆NLS-DEDD). c Representative flow cytometry biaxial plots of KTB-21 cells showing EdU incorporation with or without expression of cytosolic DEDD (ΔNLS-DEDD). DEST: control vector. d, Left: Representative immunofluorescence staining showing EdU incorporation of KTB cells with or without expression of cytosolic DEDD. 4′,6-Diamidino-2-phenylindole (DAPI): Blue; Red: EdU. Right: Quantification of EdU incorporation assay. e Representative flow cytometry biaxial plots of KTB-37 cells showing EdU incorporation with or without the expression of cytosolic DEDD after cell cycle release from nocodazole treatment (100 ng/mL) for 24 h. f Immunoblots showing cyclin profiles as well as DEDD expression of control plasmid or ∆NLS-DEDD-overexpressed normal mammary epithelial cells (KTB-37) at different time points after cells withdrawn from nocodazole treatment (100 ng/mL). g Immunoprecipitation assay showing the interaction between cytosolic DEDD and heat- shock cognate 71 kDa protein (HSC70) after overexpressing 3×-FLAG-DEL-NLS-DEDD plasmid in 293FT cells. h Representative flow cytometry biaxial plots showing EdU incorporation of HSC70-knockdown KTB-37 cells with or without the expression of cytosolic DEDD. i Western blots showing cell signaling changes of KTB cell lines with or without the treatment of HSC70 small interfering RNAs (siRNAs) for 2 days. j Bar chart showing the percentage of the immunofluorescence staining positive cells expressing either cytosolic DEDD or control vector in KTB cell lines with/without the treatment of HSC70 siRNAs for 2 days. Blue: DAPI; Red: EdU+. All quantitative data were generated from a minimum of three replicates. P values were derived from one-way analysis of variance (ANOVA) with multiple comparison test. Error bars represent means ± s.e.m.