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. 2022 Aug 1;13:4461. doi: 10.1038/s41467-022-31250-2

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© The Author(s) 2022

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 A representative TEM image of purified EVs from MDA-MB-231 cells (left), and nanoparticle tracking of purified EVs (right). Scale bar (left panel), 100 nm. b Mass-spectrometry analysis of purified EVs secreted by MDA-MB-231 cells, showing results for Annexin A2, Integrin α6, HSP90, TβRII (TGFBR2, red), TSG101, CD9, CD63 and CD81 (left). TβRII peptide identified by mass-spectrometry analysis of purified EVs from MDA-MB-231 (right panel). c Immunoblot detection of TβRII in purified EVs (E) and whole lysates (W) from different breast cancer cell lines. d TEM images of different breast cancer cell lines-derived EVs immunogold-labeled with anti-TβRII antibodies (left panel), and quantification of number of gold particles by TEM (right panel). Gold particles are depicted as black dots. Scale bar, 50 nm. e Density gradient centrifugation confirming that TβRII secreted by MDA-MB-231 cells co-fractionated with exosome markers Alix and TSG101. f FACS analysis and quantification of the percentage of TβRII positive (TβRII⁺) EVs from different breast cancer cell lines (left panel). n = 3 biological replicates per group (right panel). The percentage was referred to as the percentage of beads with TβRII⁺ EVs. g Co-localization of endogenous TβRII and Alix in MDA-MB-231 cells. Scale bars, 10 μm. h Immunoblot analysis (left) and quantification (right) of TβRII in whole cells lysate and EVs derived from control cells and TGF-β-treated cells. i Schematic diagram of biotin-labeling assay (left) and immunoblot analysis (right) measuring TβRII levels on the cell membrane and secreted EVs. j Schematic diagram (left) of ELISA to measure TβRII concentration (right) on the surface of EVs derived from 4T1 and MDA-MB-231 cells, with or without TGF-β treatment. TMB, 3,3’,5,5’-tetramethylbenzidine; SA-HRP, streptavidin-horseradish peroxidase. k ELISA of TβRII on the surface of EVs from indicated cell types. l, ELISA of TβRII on EVs isolated from MDA-MB-231 cells pre-treated with or without TGF-β (2.5 ng/ml), TβRII neutralizing antibody (10 µg/ml) or TGF-β neutralizing antibody (10 µg/ml) for 24 h as indicated. *p < 0.05 (two-tailed Student’s t test d, f, h, k, l or two-way ANOVA j). Data are analyzed of three independent experiments and shown as mean ± SD (d, f, h, j, k and l). Source data are provided as a Source Data file.