Figure 4

mTOR-HIF-α axis is critical for TNF-α/IFN-α-mediated induction of CD73 in GMSCs. GMSCs transfected with either control (si-Ctrl) or HIF-1α siRNA (si-HIF1α) were stimulated with TNF-α (100 ng/mL), IFN-α (100 ng/mL), or TNF-α/IFN-α (50 ng/mL) for 24 h (a) and 48 h (b–d). (a) Total RNA was isolated and analyzed to evaluate HIF-1α and CD73 mRNA expression using quantitative real-time PCR. (b) Whole-cell lysates prepared from GMSCs were subjected to immunoblot analysis with anti-CD73 or anti-HIF-1α antibodies. β-actin was used as the control. Relative HIF-1α and CD73 protein expressions were measured by quantifying the density of bands and normalized against β-actin using the MultiGauge software (right). (c) Exosomal CD73 protein expression in equal amounts (5 μg) was compared by immunoblot analysis with an anti-CD73 antibody. Exosome-associated 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). (d) Effect of rapamycin treatment on the expression of HIF-1α and CD73 in TNF-α, INF-α, and TNF-α/ INF-α-stimulated GMSCs. Inhibition of mammalian target of rapamycin (mTOR) activity was confirmed using a phosphorylated-mTOR antibody (p-mTOR, Se2448). Relative HIF-1α and CD73 protein expressions were measured by quantifying the density of bands and normalized against β-actin, and relative phosphorylation of mTOR were measured by quantifying the density of bands and normalized against mTOR using the MultiGauge software (right). using the MultiGauge software (b; right). *P < 0.05; **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.