Figure 5
From: Newt A1 cell-derived extracellular vesicles promote mammalian nerve growth
Fluorescently labelled A1EV generation and neuron uptake and RUNX3 expression. (A) Brightfield and confocal images of A1 cells following transfection of the CD63-GFP fusion protein plasmid. The green cytoplasmic signal shows the expression of the green fusion protein embedded in the EV membrane, with the larger, brighter green masses likely indicating the localization of EVs within multivesicular bodies as the EVs form. Scale Bar = 10 µm. (B) Confocal images of alpha acetylated tubulin-stained neurons (white) show the colocalization of the GFP-labelled A1EVs (green) with the endocytosis marker, Rab7 (red). The large blue arrows found within the images, or 10 × magnified insets, show the colocalization of A1EVs with Rab7 within the neurite, while the small yellow arrowheads show Rab7 alone. Scale Bar = 50 µm. (C) Western blots showing RUNX3 expression in A1EV or media control-treated neurons at 12 h post treatment. (D) Graphical representation of RUNX3 expression in SCG neurons, following exposure to A1EVs, with and without actinomycin D (ActD) pretreatment, as assessed by qPCR from isolated SCG neuron RNA. *** Indicate statistical significance from experimental condition without A1EVs treatment or ActD pretreatment.
