Figure 1
FGF2 is found at the outer surface of EVs secreted by dermal fibroblasts cultured in FGF2-containing medium. (A) FGF2, syntenin-1 and CD63 expression in DF cells and EV lysate (20 μg) was examined by western blot. Three isoforms of endogenous FGF2 were present in DF cells while FGF2-EVs contained the low molecular weight FGF2 isoform corresponding to recombinant FGF2. Original blots are shown in Suppl. Information. (B) Cells were cultured in the presence of His-tagged FGF2; western blot with 15 μg lysate and using anti-His antibody revealed the presence of His-FGF2 in secreted EVs. A shift in FGF2 size was also observed. Syntenin-1 was used as EV marker. Original blots are shown in Suppl. Information. (C) FGF2-EVs were loaded on a SEC column and collected fractions 8 and 9 (3E + 9p) were analyzed by western blot using antibodies for FGF2 and the EV markers CD63, CD81 and syntenin-1. Original blots are shown in Suppl. Information. For quantification, fractions were also analyzed by NTA. (D) FGF2 detection in FGF2-EVs by ELISA assay. Intact CTL- and FGF2-EVs (8E + 8p) were directly placed on FGF2 ELISA wells for external surface detection. FGF2-EVs were also treated with 0.1% triton and analyzed to detect internal FGF2. No FGF2 signal was detected in CTL-EVs. (E) FGF2 expression on FGF2-EVs was detected by flow cytometry. EVs were coupled to latex beads and labeled with FGF2 antibody before analysis by cytometry. Beads without EVs (beads) and beads labelled with goat IgG antibody (IgG) serve as negative controls. A representative plot is shown.
