Figure 2: Cell membrane-selective delivery and EV-mediated intercellular transfer of SR-lipids.

(a) Confocal fluorescent microscopic images and fluorescence quantification of HeLa cells treated with fluorophore-streptavidin (SA; red) after liposome-mediated biotin-lipid delivery for 1 h. (b) Time-dependent fluorescence change of cells after FL-mediated biotin-lipid delivery in a. (c) Confocal fluorescent microscopic images and fluorescence quantification of HeLa cells in the lower chamber of a transwell. Cells in the upper filter with 400-nm pores, treated with biotin-liposomes for 1 h, were co-incubated with fresh cells in the lower chamber for 4 h. The cells in the lower chamber were then incubated with fluorophore-SA (red) and imaged. (d) Time-dependent fluorescence change of lower-chamber cells after FL-mediated biotin-lipid delivery to the transwell filter cells in c. (e) Fluorescence quantification of HeLa cells treated with fluorophore-SA after treatment with EV-containing (+) or EV-depleted (−) supernatant collected from the cells treated with biotin-FLs. (f) Fluorescence quantification of HeLa cells in the lower chamber treated with fluorophore-SAs 4 h after FL-mediated biotin-lipid delivery to siRNA (scrambled, ARF6 or RAB 27A)-pretreated cells in the upper transwell filter. (g) Fluorescence quantification of EVs produced from the cells treated with FLs containing fluorophore-lipids after DMA treatment. (h) Nanoparticle tracking analysis of EVs produced from the cells treated with liposomes containing fluorophore-lipids. Data are means±s.e.m. (n≥10, ***P<0.001, one-way analysis of variance (ANOVA) with Tukey post test for a and c; n=3 for b and d; n=5; ***P<0.001, Student’s t-test for e,f and g; n=3, ***P<0.001, one-way ANOVA with Tukey’s post hoc test for h). Scale bar represents 20 μm.