Figure 3

Active induction of macropinocytosis by the modification of EV membranes with oligoarginine peptides. (a) Relative cellular uptake of the macropinocytosis marker FITC-dextran in the presence or absence of EVs (20 μg/ml) with or without modification by Rn-EMCS (n = 4, 8, 12: 20 μM, n = 16: 10 μM) for 3 h at 37 °C according to a flow cytometry analysis. (b,c) Relative cellular uptake of the macropinocytosis marker FITC-dextran in the presence or absence of EVs (20 μg/ml) with modification by Rn-EMCS (n = 8: 20 μM (b), n = 16: 10 μM (c)) for 1 h at 37 °C with or without the treatment with the macropinocytosis inhibitor EIPA according to a flow cytometry analysis. The data are expressed as the average (±SD) of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (d,e) Relative cellular uptake of CD63-GFP EVs (20 μg/ml) with modification by Rn-EMCS (n = 8: 20 μM (d), n = 16: 10 μM (e)) was conducted for 1 h at 37 °C with or without treatment with the macropinocytosis inhibitor EIPA according to a flow cytometry analysis. The data are expressed as the average (±SD) of three experiments. (f,g) Confocal microscope observation of CHO-K1 cells treated with EVs (20 μg/ml) modified with (g) or without (f) R16-EMCS (10 μM) for 20 min at 37 °C (enlarged pictures of Supplementary Fig. 8). Cellular staining with rhodamine-phalloidin was conducted to visualize F-actin prior to the observations.