Fig. 6.

Effects of the cortical astrocyte-derived EVs internalization on primary cortical neurons. Neurons were incubated overnight with 9–10 μg/μL of the fresh astrocyte-derived EVs. a Analysis of the neuronal mRNA expression of IL-1β in the presence of the EVs derived from the untreated and ethanol-treated (40 mM, 24 h) WT and TLR4-KO astrocytes. b Immunoblot analysis and quantification of the neuronal levels of COX-2, NFκB-p65, IL-1RI, and procaspase-1 in the presence of the EVs derived from the untreated or ethanol-treated (40 mM, 48 h) WT and TLR4-KO astrocytes. A group of untreated neurons with EVs was also added to the experiments. A representative immunoblot for each protein and their molecular weight are shown. 40 μg/μL of protein were loaded in each lane. GAPDH was used as a control loading. c Confocal microscopy analysis of the fragmented nuclei of neurons stained with Hoechst 33342 dye. 500–750 nuclei per condition were quantified. Bar graphs represent the percentage of fragmented nuclei of neurons per genotype and treatment. Data represent mean ± SEM, n = 4–5 independent experiments. *p < 0.05 compared to their respective control counterparts according to the one-way ANOVA followed by Bonferroni’s post-hoc test