Fig. 6.

Impact of ectopic lentiviral overexpression of DPP4 on EV internalization efficiency by HeLa cells. (A) Schematic of the experiment: Hela cells were infected with a control lentivirus (Myc) or a lentivirus that overexpressed DPP4 (DPP4-Myc); after selection for 20 d, EVs were then collected for analysis. (B) Western blot analysis of DPP4 levels in HeLa cells expressing in the control group (Myc lentivirus) and the DPP4 overexpressing group (DPP4-Myc virus). Proteins were collected from whole cells, from total EVs derived from the lentivirus transfected cells, and from the surface membrane of the isolated EVs. The levels of housekeeping control protein GAPDH and the EV marker CD81 were also monitored, and overall loading and membrane transfer were monitored by Ponceau S staining. (C) Left, flow cytometric analysis showing the uptake by proliferating (P) WI-38 fibroblasts after incubation with no EVs, with only PBS, and with PKH26-labeled EVs prepared from HeLa cells that were infected with the Myc lentivirus or with the DPP4-Myc lentivirus. Right, bar graphs quantify the uptake of fluorescence (MFI) after the incubations shown on the left. Data are the means ± SEM of three biological replicates; Student’s t test is used for two groups comparison. (D) A proposed model showing that S-EVs bearing DPP4 on the surface showed reduced internalization by fibroblasts (proliferating or senescent) and by HeLa cells, while they are taken up by macrophages. ***P ≤ 0.001.