Fig. 3. α_vβ₆ integrin down-regulation in exosomes inhibits recipient monocyte M2 differentiation.

Human PBMC (2.0×10⁵ cells) derived from two healthy donors were incubated for 48 hours with different concentrations of exosomes (Exo) (hi: 8 μg/mL; lo: 0.8 μg/mL) derived from PC3 cells incubated with no siRNA, a non-silencing siRNA (NS) or with one of two different siRNAs specific to β₆ mRNA (D1 and D2). Cultures were harvested at 48 hours for M2 monocyte polarization analysis by flow cytometry. (A), Contour plots depict live gating of CD14⁺ monocytes and representative expression of M2 polarization markers CD163 and CD204 in this population. Numbers indicate the percentage of gated cells. (B), There are 4 different PBMC treatment groups: untreated cells (-); NS, PBMC treated with exosomes from PC3 cells transfected with non-silencing siRNA; D1, PBMC treated with exosomes from PC3 cells transfected with a β₆-siRNA duplex designated D1; D2, PBMC treated with exosomes from PC3 cells transfected with a different β₆-siRNA designated D2. Mean fluorescence intensity (MFI). The percentages of CD14 gated CD163+/CD204+ cells in D1-hi, and D2-hi are statistically lower than NS, as assessed by Dunnett’s test. **: P > 0.01. (C), IB analysis of β₆ integrin expression in TCL and Exo derived from PC3 cells transfected with NS siRNA or with β₆ siRNA (D1 and D2). Flotilin 1 (FLOT1) was used as loading control for TCL and Exo, whereas CANX was used as loading control for TCL but not Exo.