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. 2022 May 5;20:58. doi: 10.1186/s12964-022-00863-x

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© The Author(s) 2022

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 2 — OL-EVs and OL-HSPB8-EVs uptake and chaperone activity. A–D Quantification of cellular uptake of DiI-labelled OL-EVs and OL-HSPB8-EVs by flow cytometry in human microglia C20 and primary mixed neural cultures. The data presents the mean fluorescent intensity and are shown as mean ± SEM (n = 3 biological replicates per group. Green peaks show live cells stimulated with DiI-labelled EVs and/or activated with TNFα/IL1β while grey peaks show the non-stimulated, non-activated live cells E Relative qPCR expression of HSPB8 in human microglia C20 cell line stimulated with OL-EVs or OL-HSPB8-EVs either non-activated or TNFα/IL1β activated. Data were analyzed using stable reference genes, and the fold changes were plotted as the mean ± SEM and normalized to the non-stimulated control cells (n = 3 biological replicates per group). F–H Western blotting analysis of SQSTM1/P62 and ubiquitinated proteins in HeLa HSPB8-KO cells in the soluble (non-aggregated) and insoluble fraction (aggregated). The cells were either OL-EVs, OL-HSPB8-EVs stimulated or non-stimulated and exposed to a 90 min heat shock at 42 °C. Protein levels were calculated from two independent experiments and normalized to β-actin. Band intensities were determined by quantifying the mean pixel gray values using the ImageJ software. Data are presented as mean ± SEM. One-way ANOVA was used in GraphPad to determine statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)