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. 2020 Jul 22;6(30):eaba3688. doi: 10.1126/sciadv.aba3688

Fig. 2. Extracellular ADA2 and dAdo are paracrine modulators of IFNβ.

Fig. 2

(A) ADA2 mRNA levels, measured by qRT-PCR, in primary endothelial cells and U937 monocytic cells (n = 3 technical replicates). (B) Secreted ADA2 protein, measured by ELISA, in primary endothelial cells and U937 monocytic cells (n = 3 technical replicates). (C) Secreted ADA2 protein, assessed by Western blotting of serum-free cell-conditioned supernatants, from HUVEC and U937 monocytic cells. (D) ADA activity, measured by de novo conversion of 1 mM isotopically labeled dAdo to dIno in whole-cell lysates (intracellular) or cell-conditioned supernatants (extracellular) from siControl, siADA1-, or siADA2-transfected HUVEC. Values are normalized to cell number and protein concentration (n = 5 technical replicates). (E) Expression levels of IRF3-driven or IFN-β–driven genes, measured by qRT-PCR, and extracellular ADA activity, measured by de novo conversion of 1 mM isotopically labeled dAdo to dIno in cell-conditioned supernatants, from siControl or siADA2-transfected HUVEC supplemented with rADA1 or rADA2 and pretreated with vehicle or pentostatin (10 μM for 30 min). Activity values are normalized to cell number and protein concentration (n = 5 technical replicates). (F) IFN-β mRNA, measured by qRT-PCR, in U937 monocytic cells supplemented with adenosine (Ado) or deoxyadenosine (dAdo) (100 μM for 48 hours) (n = 3 technical replicates). (G) IFN-β mRNA levels, measured by qRT-PCR, in U937 cocultured in Transwell inserts with siControl or siADA2-treated HUVEC for 48 hours (n = 3 technical replicates). All results were replicated in three independent experiments. Values are presented as means ± SD. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. HDVEC, dermal microvascular endothelial cells; HBVEC, brain microvascular endothelial cells; HKVEC, kidney microvascular endothelial cells.