Supplementary Figure 11: Human and bovine vitreous extracellular vesicles (EVs) transfer endogenous RNA into cultured cells.

a, Representative confocal photomicrograph images of a human retinal pigment epithelial cells (ARPE-19) after 24 h treatment with a bolus of bovine vitreous EVs that were pre-labeled with the nucleic acid stain acridine orange (AO). Images show uptake of labelled EV-RNA in ARPE-19 cells (left and right panels, AO, green). Nuclei are labeled (middle and right panels, Hoechst, purple), and a merged image (right panel) show transfection of ARPE-19 cells, with AO signal (green) in the cytoplasm. b, Graphical representation of mean ± standard deviation transfection efficiency (% of cells transfected) for ARPE-19 cells treated with bovine vitreous EVs for 3, 6, or 24 h (n = 3 cell cultures, *P = 0.00002 based on two-tailed, unpaired Student’s t-tests for ARPE-19 cells treated with labelled bovine EVs for 3 h versus control (t value 237.9, df 2), P = 0.0003 for ARPE-19 cells treated with labelled bovine EVs for 6 h versus control (t value 55.9, df 2), P = 0.00005 for ARPE-19 cells treated with labelled bovine EVs for 24 h versus control (t value 147.8, df 2)). c, Representative wide-field epi-fluorescent low-power photomicrographs of ARPE-19 cells treated with a 3 h bolus of EVs that were isolated from post-mortem human vitreous and pre-labeled with AO. Images show transfection of cells (left panel, AO, green). Nuclei were marked (right panel, Hoechst, blue). d, Graphical representation of of mean ± standard deviation transfection efficiency (% of cells transfected) for ARPE-19 cells treated with human vitreous EVs (n = 3 cell cultures, *P = 0.0001 for ARPE-19 cells treated with labelled human EVs for 3 h versus controls (t value 93.5, df 2) and P = 0.0002 for ARPE-19 cells treated with labelled human EVs for 24 h versus control (t value 64.3, df 2), based on a two-tailed, unpaired Student’s t-test). Scale bars are (a) 15 µm and (c) 100 µm.