Fig. 1: Hypoxia-induced changes to the protein composition of NSCLC cell-derived sEVs.

a The morphology of isolated sEVs was assessed using transmission electron microscopy. Representative images of normoxic and hypoxic SKMES1-derived sEVs (Size bar 200 nm) also indicate clear upregulation of sEV concentration. b Western blot of sEVs from SKMES1 demonstrating the presence of sEV heat shock protein 70 (HSP70), flotillin-1 (FLOT1), and CD63 antigen (CD63) and the absence of the cell marker calnexin (CANX). c Nanoparticle analysis using tunable resistive pulse sensing (TRPS) of sEVs isolated from SKMES1 under normoxic and hypoxic conditions demonstrating the majority of sEVs have a size range between 30 and 150 nm, and that hypoxia increases sEV secretion. d Volcano plot of quantitative mass spectrometry identifying 426 proteins that are upregulated in SKMES1 sEVs (FDR < 0.005; n = 4 independent replicates). e, Heatmap demonstrating quantitative mass spectrometry identification of 6 proteins to be significantly upregulated in hypoxic sEVs derived from SKMES1 cells. f, g Mass spectrometry results were confirmed using western blot of GANAB and VCP (f), and ELISA for MAC2BP, PSMA2, THBS1, and TNC in H23, H358, H1975, and SKMES1 NSCLC cell lines (g) (▲ – H23, ● – H358, ♦ – H1975, ■ – SKMES1). *p < 0.05, **p < 0.01.