Fig. 4
miR-298-5p and miR-100-5p enter microglia, co-localize to their endosomes, and bind directly to human TLR8. a C57BL/6 microglia were incubated with 40 μg/ml pHrodo Red Dextran serving as endosomal marker for 20 min. Subsequently, microglia were exposed to 5 μg/ml of Alexa488-labeled miR-298-5p, Alexa488-labeled miR-100-5p, or Alexa488-labeled let-7b-5p for 4 h. Cells were then fixed and stained with DAPI. b In parallel, microglia exposed to Alexa488-labeled miR-298-5p or Alexa488-labeled miR-100-5p, as described above, were fixed and immunolabeled with TLR7 antibody. (a, b) Cells were analyzed by confocal microscopy with sequential analysis. Representative images of microglia incubated with the indicated fluorescent miRNAs (488 nm, green), stained with DAPI (405 nm, blue), and pHrodo Red Dextran or TLR7 (552 nm, red) are depicted. Green lines indicate region of interest (ROI); scale bar, 10 μm. Representative 3D-vertical slice images of microglia show co-localization of miRNAs and endosomes through different cellular levels (left panel). Diagrams depict fluorescence intensities of the marked ROI in microglia for the sequential analysis used (DAPI: blue line; pHrodo Red Dextran/TLR7: red line; fluorescent miRNA: green line, right panel). (c) Binding affinity measurements of purified polyhistidine-tagged human TLR8 protein and synthetic miRNAs, as indicated, using microscale thermophoresis (MST). TLR8-RNA interaction was monitored by titrating indicated miRNAs from 500 μM to 30 nM against 50 nM RED-tris-NTA-labeled hTLR8 protein and measured with the NanoTemper Monolith NT.115 MST device. Kd values were calculated from dose response curves, which were generated from titration experiments. Data are expressed as mean ± SD, n = 4