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. 2023 Jul 17;60(11):6441–6465. doi: 10.1007/s12035-023-03456-y

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© The Author(s) 2023

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 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/.

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Fig. 7 — Impact of HBMEC-derived EVs on synaptic protein expression in NPCs. Non-transfected HBMEC were treated with HIV and/or Aβ and EVs were isolated as in Fig. 3. Then, human NPCs were exposed to HBMEC-derived EVs for 24 h, with selected cultures additionally treated with 2 µM PAI039 (P) as in Fig. 6. A synaptophysin (green; arrow heads) and PSD95 (red; arrows) immunoreactivity as imaged by confocal microscopy. DAPI staining (blue) visualizes the NPC nuclei. The combined z-stack images with maximum intensity projection are representative from three experiments. Scale bar: 10 μm. Individual panels are shown in Supplementary Fig. 2. B Total intensity of synaptophysin (left graph) and PSD95 (right graph) immunoreactivity as quantified from the confocal images. Values are mean ± SEM, n = 7. One-, two- and three-way ANOVA with Šídák's multiple comparisons tests. C Example of a brightfield confocal image with random identical rectangular areas on NPC projections. D Synaptophysin (green) and PSD95 (red) immunoreactivity on representative confocal images of NPC projections. Scale bar: 2 μm. E Intensity of synaptophysin and PSD95 immunoreactivity as quantified from random projection areas. Values are mean ± SEM, n = 160–180. One-, two- and three-way ANOVA with Šídák's and Tukey’s multiple comparisons tests. *Statistically significant at p < 0.05, ****p < 0.0001

Fig. 7 — Impact of HBMEC-derived EVs on synaptic protein expression in NPCs. Non-transfected HBMEC were treated with HIV and/or Aβ and EVs were isolated as in Fig. 3. Then, human NPCs were exposed to HBMEC-derived EVs for 24 h, with selected cultures additionally treated with 2 µM PAI039 (P) as in Fig. 6. A synaptophysin (green; arrow heads) and PSD95 (red; arrows) immunoreactivity as imaged by confocal microscopy. DAPI staining (blue) visualizes the NPC nuclei. The combined z-stack images with maximum intensity projection are representative from three experiments. Scale bar: 10 μm. Individual panels are shown in Supplementary Fig. 2. B Total intensity of synaptophysin (left graph) and PSD95 (right graph) immunoreactivity as quantified from the confocal images. Values are mean ± SEM, n = 7. One-, two- and three-way ANOVA with Šídák's multiple comparisons tests. C Example of a brightfield confocal image with random identical rectangular areas on NPC projections. D Synaptophysin (green) and PSD95 (red) immunoreactivity on representative confocal images of NPC projections. Scale bar: 2 μm. E Intensity of synaptophysin and PSD95 immunoreactivity as quantified from random projection areas. Values are mean ± SEM, n = 160–180. One-, two- and three-way ANOVA with Šídák's and Tukey’s multiple comparisons tests. *Statistically significant at p < 0.05, ****p < 0.0001