Fig. 2

Extracellular miR-124-5p and miR-92a-1-5p enter neurons and co-localize to endosomes. (a) C57BL/6 cortical neurons were incubated with Alexa488-labeled 10 µg/mL of miR-92a-1-5p, Alexa488-labeled miR-124-5p (both), or unconjugated Alexa488 ester. Cells stained with Hoechst and immunolabeled with Neurofilament or NeuN antibodies were analyzed by confocal microscopy. Representative images of neurons exposed to miRNAs for 8 h. (b) Mean fluorescence intensity of Alexa-488-labeled miRNAs and ester within neurons, depicted as time course graph. Lines and error bars represent mean ± SEM (n = 3–4). (c) Representative images of neurons incubated with pHrodo Red (20 µg/mL) and fluorescent miRNAs (10 µg/mL), as indicated, for 4 h. Cells were stained with Hoechst and analyzed by confocal microscopy with sequential analysis. Arrow heads indicate fluorescent miRNAs co-localising to pHrodo Red Dextran. White lines indicate regions of interest (ROI). (d) Line profiles depicting fluorescence intensities along the marked ROIs. (e) Representative images of neurons treated as described in (c), set up for confocal time-lapse imaging for 4 h. (f) Kinetic curves depicting the fluorescence intensity of fluorescent miRNAs or ester and pHrodo Red within endosomes over the 4 h imaging period. (g) Neurons described above treated in parallel with DMSO or Dynasore (200 µM) for 4 h and analyzed by confocal microscopy. (h) Fluorescence intensity of fluorescent miRNAs and pHrodo Red within endosomes of neurons described above. Bars represent mean ± SEM (n = 5). **P < 0.01; ***P < 0.001, unpaired t-test, compared to the corresponding DMSO group. Scale bar, 20 μm