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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. 3 — OL-EVs and OL-HSPB8-EVs stimulation activate autophagy in human microglia and primary mixed neural cells. A, B Western blotting analysis of LC3B-II and BAG3 in OL-EVs and OL-HSPB8-EVs. Protein levels were calculated from two independent experiments and normalized to the EVs marker flotillin-1. Band intensities were determined by quantifying the mean pixel gray values using the ImageJ software. Data are presented as mean ± SEM. C, D Immunofluorescence staining of human microglial C20 cells stimulated with OL-EVs or OL-HSPB8-EVs. Cells were treated with chloroquine to inhibit lysosomal degradation. Rapamycin was used as a positive control. Representative image showing LC3B (green), the DAPI nucleus stain (blue), and merged images. CellProfiler was used to quantify the LC3B punctate structures (LC3B, mean fluorescence intensity) (n = 4 biological replicates, 15 + cells per experiment). Data are expressed as mean ± SEM, ****P < 0.0001. E–H Flow cytometry monitoring CYTO-ID autophagic flux in chloroquine treated human microglia C20 cell line and a primary mixed neural culture using the CYTO-ID green detection dye, selectively labeling autophagic vacuoles. Data are presented as mean fluorescent intensities ± SEM (n = 3 biological replicates per group), Green peaks represent TNFα/IL1β activated and/or EV stimulated cells, while the grey peaks show cells of non-activated and non-stimulated control cells. GraphPad was used to determine significance (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). I, J Ultrastructural analysis of autophagic vesicles by transmission electron microscopy of C20 cells stimulated with OL-EVs or OL-HSPB8-EVs. Lysosomal degradation was blocked using chloroquine. Rapamycin was used as a positive control. The total number of autophagic vesicles per field of view were counted and expressed per cytoplasmic area covered (scale bar = 1 µm). Data are shown as mean ± SEM. GraphPad was used for statistical analysis by using one-way ANOVA (**P < 0.01)