Adenosine 5 monophosphate disodium salt

For cell-based ¹³C₆ metabolite labeling analysis, around 90% confluent H1299 cells were washed by glucose-free culture medium (Gibco, 11966-025) and cultured in medium containing ¹³C₆-glucose (10 mM) (Cambridge Isotope Laboratories, CLM-1396) for 30 min. High-resolution LC–MS/MS was used for analyzing the levels of ¹³C₆-labeled intracellular metabolites^{11 (link)}.
For label-free xenograft metabolite analysis, the xenograft tissue samples were homogenized in 150 μl of extraction solution (half methanol and half water) with 250 ng ml⁻¹ 4-nitrobenzoic acid and debrisoquine as internal standard for negative and positive modes, respectively, followed by adding 150 μl of acetonitrile. After incubation for 30 min at −20 °C, the samples were centrifuged at a speed of 16,000g for 30 min at 4 °C. The standards—namely inosine 5′-monophosphate disodium salt (IMP, I4625), adenosine 5′-monophosphate disodium salt (AMP, 01930), guanosine 5′-monophosphate disodium salt (GMP, G8377), uridine 5′-monophosphate disodium salt (UMP, U6375) and cytidine 5′-monophosphate disodium salt (CMP, C1131)—were obtained from Sigma-Aldrich. The samples were transferred to MS vials for LC–MS analysis by QTRAP 7500 System (SCIEX). Data were acquired and normalized to the internal standards and then analyzed with SCIEX OS software.

Kyoto Green was synthesized following the procedure described in Ojida et al.^{23 (link)}. Magnesium chloride (MgCl₂), zinc nitrate (Zn(NO₃)₂), HEPES minimum 99.5% titration (C₈H₁₈N₂O₄S), p-nitrophynyl thymidine 5′-monophosphate (pNph-5′-TMP), sodium pyrophosphate decahydrate (NaPPi), adenosine 5′-triphosphate disodium salt (ATP), and adenosine 5′-monophosphate disodium salt (AMP) were products of Sigma-Aldrich (St. Louis, MO, USA) or Merck (Darmstadt, Germany). Cordycepin, uridine, cytidine, inosine, thymidine, kaempferol, 2′-deoxyadenosine, 2′-deoxyinosine, 2′-deoxyuridine, and 2′-deoxyguanosine were purchased from Fujifilm Wako Pure Chemical Corporation (Japan). Myricetin, quercetin dihydrate and guanosine were commercially available from Alfa Aesar (UK). Recombinant human ENPP1 was purchased from R&D systems (Minneapolis, USA).

Amino acid standard mix containing alanine (Ala), glycine (Gly), valine (Val), leucine (Leu), isoleucine (Ile), threonine (Thr), serine (Ser), proline (Pro), aspartic acid (Asp), methionine (Met), 4-hydroxyproline (Hyp), glutamic acid (Glu), phenylalanine (Phe), lysine (Lys), histidine (His), tyrosine (Tyr), tryptophan (Trp), and cystine (C–C) was purchased from Phenomenex, Torrance, USA. Solvents used were HPLC grade and purchased from Sigma-Aldrich, Copenhagen, Denmark.
5′-mononucleotide standards including adenosine 5′-monophosphate disodium salt (AMP, ≥ 99.0%), guanosine 5′- monophosphate disodium salt hydrate (GMP, ≥ 99%), and inosine 5′-monophosphate disodium salt hydrate (IMP, ≥ 99.0%) were obtained from Sigma-Aldrich, Copenhagen, Denmark. Xanthosine 5′-monophosphate disodium salt (XMP, ≥ 98%) and uridine 5′-monophosphate disodium salt (UMP, ≥ 99%) were obtained from Carbosynth, Compton, UK.
Ultrapure Milli-Q water (Milli-Q system, Millipore, Bedford, MA, USA) was used throughout the study.