Fig. 5
Hypoxia alters the cargo of EVs. A) Multiplex phenotypic analysis by flow cytometry (expressed as the median fluorescence intensity) showed the differential expression pattern of membrane proteins on EVs collected from CAFs, PMNs, HUVECs and HBEC-KRASV12high cells (n = 5). B) Relative c-Myc mRNA expression levels (left) and protein content (WB, right) in fibroblasts under normoxic or hypoxic (2% O2) conditions (n = 5). C) Quantification of c-Myc protein content in EVs released by normoxic and hypoxic fibroblasts (n = 5). D) Representative images and analysis results of HBEC-KRASV12high colony formation in VitroGel after treatment with normoxic and hypoxic fibroblast-derived EVs. E) Relative mir-126 expression levels in HUVECs exposed to normoxia or hypoxia (2% O2) (n = 3). F) Quantification of miR-126 in EVs released by normoxic and hypoxic HUVECs (n = 3). G) Analysis of tube formation and endothelial cell activation after treatment with EVs derived from HUVECs under normoxic or hypoxic conditions (n = 5). H) Relative expression levels of intracellular miR-320 in PMNs cultured for 24 h under hypoxic (2% O2) or normoxic conditions (left) and in EVs (right) (n = 5). I) Analysis of miRNA-320 and M2 polarization gene mRNA expression levels (left) and CD163/CD206 expression levels by flow cytometry (right) in macrophages treated with EVs released from PMNs cultured under normoxic and hypoxic conditions (n = 5). Untreated cells (NT) were used as a control. *p < 0.05 versus controls. The data are expressed as the mean ± S.E.M. values