Fig. 4. Loss of ADA2 drives intracellular dAdo catabolism and accumulation of dIno.

(A) Intracellular metabolic pathways of purine degradation [ADA1 (protein); PNP (protein); and HGPRT (protein), hypoxanthine phosphoribosyltransferase] and purine salvage [ADK (protein), adenosine kinase; dCK (protein), deoxycytidine kinase; and 5′-NT, 5′-nucleotidase]. (B) Relative levels and concentrations of small-molecule polar metabolites measured by LC-MS/MS quantification of siControl or siADA2-transfected HUVEC (n = 5 biological replicates). (C) De novo accumulation of ¹⁵N-dAdo–labeled dIno measured by LC-MS/MS quantification of siControl or siADA2-treated HUVEC (n = 5 biological replicates). (D) IFN-β mRNA levels, measured by qRT-PCR, in HUVEC transfected with siRNAs targeting ADA2 and ADA1, PNP, HGPRT (protein), or dCK. (D) IFN-β mRNA levels, measured by qRT-PCR, in HUVEC supplemented with dIno (500 μM for 24 hours) or (E) pretreated with the PNP inhibitor 9-deazaguanine (100 μM for 30 min) before dIno supplementation (500 μM for 24 hours) (n = 3 technical replicates). (F) Flux analysis of the methionine/SAM cycle, measured by LC-MS/MS quantification of U-¹³C methionine–labeled methionine, SAM, and SAH, in siControl or siADA2-treated HUVEC (n = 5 biological replicates). (G) Methionine adenosyltransferase (MAT) enzyme activity, measured by LC-MS/MS quantification of SAM, in HUVEC lysates supplemented with nucleosides (n = 3 technical replicates). (H) IFN-β mRNA levels, measured by qRT-PCR, in HUVEC pretreated with cycloleucine (MAT inhibitor, 20 mM for 30 min) or 5-azacytidine (DNMT inhibitor, 500 nM for 30 min) (n = 3 technical replicates). (I) De novo DNA methylation, measured by LC-MS/MS quantification of U-¹³C-methionine labeled 2′-deoxy-5-methylcytidine in siControl, siADA2-, or siDNMT1-treated HUVEC (n = 3 biological replicates). Values are represented as the percentage of total label incorporation. All results were replicated in three independent experiments. Values are presented as means ± SD. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. ADP, adenosine diphosphate; IMP, inosine monophosphate; THF, tetrahydrofolate; MTR, 5-methyltetrahydrofolate-homocysteine methyltransferase or methionine synthase.