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. 2021 Apr 23;9:634355. doi: 10.3389/fcell.2021.634355

FIGURE 5.

FIGURE 5

sEVs derived from pre-miR-146a-treated mSOD1 astrocytes show enriched content in miR-146a, but not those from VS-treated cells. Astrocytes were isolated from the cortex of SOD1-G93A (mSOD1) and wild type (WT) mice pups at 7-day-old and cultured for 13 days in vitro. Transfection with pre-miR-146a and treatment with dipeptidyl vinyl sulfone (VS) were performed in mSOD1 astrocytes. Small extracellular vesicles (sEVs) were isolated from the secretome of astrocytes by differential ultracentrifugation. (A) Results from one blot shows the expression of sEV markers (Alix and Flotillin-1). (B) Representative images obtained by transmission electron microscopy of sEVs show their cup shape morphology. Results from (C) number of sEVs per cell, (D) concentration (sEVs number/mL), and (E) size distribution derived from Nanoparticle Tracking Analysis using NanoSight. (F,G) RT-qPCR analysis of miRNA(miR)-146a expression in sEVs was performed. Spike and SNORD were used as endogenous controls. Results are mean (±SEM) fold change vs. sEVs-derived WT astrocytes from at least three independent experiments. *p < 0.05 vs. sEVs from WT astrocytes; ##p < 0.01 vs. sEVs from untreated mSOD1 astrocytes. One-way ANOVA followed by Bonferroni post hoc test was used.