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. 2021 Oct 13;9:735888. doi: 10.3389/fcell.2021.735888

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Copyright © 2021 Torres, Barrera, Varas-Godoy, Arancibia and Inestrosa.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Nanoparticle tracking analyses of exosomes secreted by control and Wnt-C59 treated HT-22 cells and the effect of Wnt-C59 on the content of exosomal markers in the P100 fraction. (A) Representative Nanoparticle tracking analyses show a mean peak for both control and Wnt-C59 samples. (B) Abundance analysis shows that 80% of the vesicles have a diameter of <200 nm (Control: 77.88 ± 2.41%, Wnt-C59: 79.20 ± 5.22; n = 4) and a 20% >200 nm (Control: 22.12 ± 2.41%, Wnt-C59: 20.83 ± 5.22; n = 4). (C) Concentration of particles. Shapiro–Wilk and KS tests showed that the data conformed to a normal distribution. Subsequently, the result of the Student unpaired t-test was p = 0.8272 with values of 2.3 × 10⁹ ± 7.6 × 10⁸ (control; n = 7) and 1.9 × 10⁹ ± 8.3 × 10⁸ (Wnt-C59; n = 7). (D) Mean distribution size of the total extracellular vesicles. Shapiro–Wilk and KS tests showed that the data conformed to a normal distribution. Subsequently, the result of the Student unpaired t-test was p = 0.7602 with values of 166.6 ± 2.5 (control; n = 7) and 164.8 ± 3.9 nm (Wnt-C59; n = 7). (E) Mode distribution size. As in (D), values conform with a normal distribution. The student unpaired t-test result was p = 0.7824 with values of 128.5 ± 0.8 nm (control; n = 7) and 128.1 ± 0.4 nm (Wnt-C59; n = 7). n value means the number of independent cell culture preparations. (F) Representative western blot analysis of the protein content of TSG101, Alix, Flotillin-1, CD63, NCAM-L1 in P100 from control and C59 treated HT-22 cells. GM130 purity control is absent in the P100 fraction. (G) Relative protein expression levels of exosomal markers. The staining intensity of each band derived from control P100 was assigned a value of 100%, and the value obtained in the P100 from Wnt-C59 treatment was compared with its respective control. Flotillin-1 was the only exosomal marker whose expression level underwent a significant decrease in exosomes secreted by WntC59 treated HT-22 cells. TSG101 (Control: 100%, Wnt-C59: 132 ± 81.3%; n = 3; p = 0.700); Alix (Control: 100%, Wnt-C59: 87 ± 45.9%; n = 3; p = 0.6496); Flotillin-1 (Control: 100%, Wnt-C59: 58 ± 8.7%; n = 3; p = 0.0011); CD63 (100%, Wnt-C59: 76.3 ± 20.6; n = 3; p = 0.0952); NCAM-L1 (Control: 100%, Wnt-C59: 161 ± 157.9; n = 3; p = 0.3831). Mann–Whitney non-parametric t-student test (ns, not significant; ***p < 0.005).