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. 2022 Jun 8;29(11):1662–1675. doi: 10.1038/s41417-022-00485-y

Fig. 4. Breast tumor cells released exosome-like vesicles with heterogeneously expressed ST6GAL1.

Fig. 4

4T1.2 cells were transfected with shControl or ShST6GAL1 #1 for 24 h, as mentioned above. Cells were cultured in the serum-free conditioned medium for another 48 h. A An equal amount of proteins from cell extracts (left panels) and exosome-like particles (right panels) were used for Western blot analysis with the indicated antibodies. Representative blots (N = 3) were shown. ST6GAL1 from cell lysates versus exosomes was analyzed in the same gel/membrane to compare their molecular size and presented in separate figures. Equal amounts of proteins from exosome fractions were used for α2,6 N-Linked activity (ST6GAL1) (B, upper panel) and α2,3 N-Linked activity (ST3GAL6)(B, lower panel) assays. Specific activity was calculated as fmol/min/µg specific product, plotted as fold activity, n = 3, data are means ± s.e., Student’s t-test, p < 0.001. NS; not significant. C, D Size distributions by nanoparticle tracking analysis (NTA) and images of exosome-like particles, which are screenshots from recorded videos of EVs when characterized by NTA. Exosome-like particles were isolated from shControl (C) and shST6GAL1 (D) transfected 4T1.2 cell culture-conditioned medium (100× dilution) and were examined by NTA. (C, D; right panel, respectively) Negative stain transmission electron microscopy (TEM) imaging of exosome-like particles. Representative images are shown. Scale bars: 100 nm.