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

FIGURE 8.

FIGURE 8

Secretome from pre-miR-146a-treated mSOD1 astrocytes translates into microglial miR-146 increase and regulation of cell activation by the untreated secretome, while also prevents cell demise similarly to VS modulation. Astrocytes (Ast) were isolated from the cortex of SOD1-G93A (mSOD1) and wild type (WT) mice with 7-day-old and cultured for 13 days in vitro. Transfection with pre-miR-146a or treatment with dipeptidyl vinyl sulfone (VS) was performed in mSOD1 astrocytes. Secretome was isolated and incubated in naïve N9 microglia for 24 h. Expression of (A) miRNA(miR)-146a, (B) fibroblast growth factor receptor 3 (FGFR3), (E) inducible nitric oxide synthase (iNOS) and (F) tumor necrosis alpha (TNF-α) in microglia was assessed by RT-qPCR. SNORD110 was used as reference gene for (A) analysis and β-actin for (B,E,F) analysis. (C) Early apoptotic (Annexin V-PE positive and 7-AAD negative), and (D) late apoptotic/necrotic cells (Annexin V-PE and 7-AAD positive) were assessed by Guava Nexin® Reagent in the microglia after secretome interaction. Results are mean (±SEM) fold change vs. MG + secretome from WT astrocytes from at least three independent experiments. *p < 0.05 and **p < 0.01 vs. MG + secretome from WT astrocytes; ##p < 0.01, ###p < 0.001, and ####p < 0.0001 vs. MG + secretome from mSOD1 astrocytes. One-way ANOVA followed by Bonferroni post hoc test was used.