Journal: Frontiers in Pharmacology
Article Title: Amanita muscaria extract potentiates production of proinflammatory cytokines by dsRNA-activated human microglia
doi: 10.3389/fphar.2023.1102465
Figure Lengend Snippet: AME-1 upregulates TLR3 expression. (A) HMC3 cells were treated with AME-1 (50–5,000 μg/mL) for 3 h, RNA was extracted and analyzed via RT-qPCR. Results are presented as fold-gene expression relative to untreated cells and statistics were calculated using One-Way ANOVA with Dunnett’s multiple comparison post hoc analysis relative to fold-change in GAPDH expression: p ≤ 0.01 (**). Data presented as mean ± SEM for n = 3. HMC3 were treated with AME-1 (50–5,000 µg/mL), LPS (1 µg/m) or poly(I:C) (0.5 µg/mL) for 24 h, the cells were lysed and analyzed via Western blot. (B) TLR3 and IRF3 protein expression and (E, F) corresponding densitometry analysis. (C, D) MDA5 and RIG-I protein expression and (G, H) corresponding densitometry analysis. Densitometry analysis was performed on three blots on the indicated bands and statistical significance was calculated using one-way ANOVA with Tukey post hoc analysis.
Article Snippet: The membrane was subsequently blocked for 1 h at room temperature with 5% skim milk in TBS-T followed by incubation for1hr with 0.4 μg/mL of mouse anti-human TLR3 (sc-32232, Santa Cruz Biotechnology, Dallas, TX, United States), 0.5 μg/mL of mouse anti-human IRF3 (550428, BD biosciences), 2.5 μg/mL of rabbit anti-human MDA5 (33H12L34, Invitrogen), 1 μg/mL rabbit anti-human RIG-1 (PA510297, Invitrogen), or 1 μg/mL of mouse anti-β-actin loading control (PA1-183, Invitrogen) in 3% BSA-TBS-T, and finally incubated for 1 h with goat anti-mouse IRDye 680 (Li-Cor, Lincoln, NE, United States) or goat anti-rabbit IRDye 800 (Li-Cor) diluted 1:15,000 in 3% BSA-TBS-T.
Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Comparison, Western Blot
