Fig 1.
Characterization of extracellular vesicles.
A: Size distribution, evaluated by nanosight, indicates that sizes are compatible with exosomes. B: Electron microscopy revealed the “donut-like” appearance characteristic of exosomes. C: Western blot of exosomes samples and cell lysates (CL) to confirm the presence of classical exosome markers (ALIX, TSG101) and the absence of endoplasmic reticulum contamination (Calnexin CXN).
Fig 2.
To determine the exosome uptake, BICR18 cells (above) stained with PKH67 (green) were incubated 2 h with 3 μg/ml of PKH26 stained exosomes (red) obtained from THP1 cells.
In parallel experiments, THP1 cells (below) were incubated with exosomes obtained from BICR18 cells. In both cases, exosome uptake can be observed.
Fig 3.
A) Microscopic images showing the BICR18 migratory pattern in control, co-cultured (CC) with THP1 macrophages or treated with 3 μg/ml THP1-exosomes. Both Co-culture and exosomes increases the cellular migration of BICR18 cells. B) The inhibition of exosome secretion with GW4869 prevented the increase of migration induced by coculture. * = p < 0.05 vs Control + p<0.05 vs untreated.
Fig 4.
mRNA expression of M1 (IL1β) and M2 (MRC1 and IL-10) markers in THP1 macrophages cocultured 24 h with BICR18 cells (c.c.) or treated with 3 μg/ml exosomes (Exo.) obtained from BICR18 cells.
* = p < 0.05 vs Control; + = p < 0.05 vs c.c.
Fig 5.
mRNA expression of PD-L1 in BICR18 or THP1 cells after 24 h co-culture or treatment with 3 μg/ml of exosomes obtained from the corresponding cells.
* = p < 0.05 vs Control; + = p < 0.05 vs c.c.
Fig 6.
(A) Immunofluorescent analysis of STAT3 subcellular location in BICR18 cells. In control cells STAT3 was found mainly in the cytoplasm, although also it was also detected in the nuclei. Treatment with exosomes obtained from THP1 macrophages promoted the activation and almost total nuclear translocation of STAT3. This activations was prevented by treatment with STAT3 inhibitor 5,15-DPP. (B) STAT3 inhibition with 5,15-DPP results in complete inhibition of exosome-induced SOCS3. For PD-L1 the inhibition is even more marked. * = p < 0.05 vs Control; + = p < 0.05 vs Exo.