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. 2020 Feb 13;9(2):16. doi: 10.1038/s41389-020-0204-5

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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. 6 — a Comparison of overall survival between patients bearing grade 3 tumors expressing levels of TNS1 above (high) or below (low) the mean level for the entire patient cohort. Survival curves were analyzed via a log-rank test resulting in the indicated p value. b Immunoblot analyses of EVs collected from control (GFP) and TG2-overexpressing HME2 (TGM2) as well as control (shMT) and TG2-depleted (shTGM2) HME2-BM cells. Lysates from these EVs were assessed for the levels of TG2 and TNS1, and the levels of CD63 served as a loading control. c Immunoblot analyses of EVs derived from control (shscram) and Tns1-depleted (shTns1) 4T1 cells. Lysates from these EVs were assessed for the levels of TG2 and Tns1, and the levels of CD63 served as a loading control. d The fibrillar FN-specific (FN3) antibody was used in conjunction with immuno-electron microscopy to analyze nonpermeabilized EVs derived from control (shscram) and Tns1-depleted (shTns1) 4T1 cells. e Control (shscram) and Tns1-depleted (shTns1) 4T1 cells were grown under single-cell 3D culture conditions. Cellular outgrowth was quantified by bioluminescence at Day 8. Data are normalized to the plated values and are the mean ± SD of three independent analyses resulting in the indicated p value. f Control (shscram) and Tns1-depleted (shTns1) 4T1 cells were engrafted onto the mammary fat pad, and the resultant primary tumors were removed and weighed upon necropsy. g Quantification of bioluminescent radiance from the thoracic regions of interest (ROIs) at the indicated time points. h Upon necropsy, the numbers of pulmonary metastatic nodules were quantified. (Inset) Corresponding gross anatomical views of lungs from control (shscram) and Tns1-depleted (shTns1) 4T1 tumor-bearing mice. Data in f–h are mean ± SE values from five mice per group resulting in no significance (n.s.) or the indicated p values.