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Figure 1

From: Extracellular vesicles derived from GMSCs stimulated with TNF-α and IFN-α promote M2 macrophage polarization via enhanced CD73 and CD5L expression

Figure 1

Effect of TNF-α/IFN-α stimulation on EVs and cellular expression of CD73 in GMSCs. GMSCs were stimulated with TNF-α (100 ng/mL), IFN-α (100 ng/mL), and TNF-α/IFN-α (50 ng/mL each) for 24 and 48 h. (a) Expression of CD73 mRNA was determined by quantitative real-time PCR. GAPDH was used as an internal control. (b) Whole-cell lysates prepared from GMSCs were subjected to western blot analysis using anti-CD73 antibody. β-Actin was used as a loading control for the cytosolic proteins (upper). Relative CD73 protein expressions were measured by quantifying the density of bands and normalized against β-actin using the MultiGauge software (lower). (c) Cell surface expression of CD73 was evaluated by flow cytometry. (d) Representative transmission electron micrographs (TEM) of EVs from GMSC with or without (EVs-Ctrl) priming of TNF-α (EVs-TNF), IFN-α (EVs-IFN), and TNF-α/IFN-α (EVs-TNF/IFN). Scale bar = 100 nm. (e) Size distribution of EVs-Ctrl, EVs-TNF, EVs-IFN and EVs-TNF/IFN were measured by NanoSight analyzer. (f) CD73 protein expression in EVs was compared in equal amounts of protein (5 μg) (upper). Exosomal positive marker of CD9 and CD81 proteins were used as loading controls. Relative CD73 protein expressions were measured by quantifying the density of bands and normalized against CD81 using the MultiGauge software (lower). **P < 0.01; ***P < 0.001. Error bars represent mean ± SD, n = 3. The significance of differences between groups was determined using one-way Tukey's test. Full blots are presented in Supplementary materials.

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