Figure 3
EV-bound FGF2 is protected from degradation. (A) Effect of EV binding on FGF2 thermal stability. FGF2 (10 ng/ml), alone or after incubation with EVs (8E + 8p) from different cell types for 1 h on ice, was placed at 37 °C for 24 h to challenge FGF2 thermal stability. Residual FGF2 activity was determined by (a) proliferation assay by BrDU incorporation and (b) ELISA assay to measure ESM-1 secretion. EVs from DF, MSC, HEK cells were tested. Results are mean ± SE of four independent experiments. Statistical significance was determined by one-way ANOVA (Dunnett’s multiple test), *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001. (B) Effect of EV binding on FGF2 degradation by trypsin. FGF2 (10 ng), alone or after incubation with EVs from DF, MSC or HEK cells (8E + 8p) for 1 h on ice, was held at 37 °C with trypsin/EDTA (0.05%, diluted 1:2 v/v in 25 μl) for different time periods before loading on western blot to assess residual FGF2 protein. (a) Representative blot with CTL-EVs (b) Representative blot and quantification of FGF2 signal obtained after 10 min incubation with EVs from DF, MSC or HEK cells. Results are mean ± SE of 3–5 independent experiments. Statistical significance was determined by one-way ANOVA (Dunnett’s multiple test), *< 0.05. Original blots are shown in Suppl. Information.
