Guanosine

Creatinine, hippuric acid, uric acid, hypoxanthine, xanthine, myoinositol, cis-aconitic acid, indoxyl sulfate and aldosterone were obtained from the National Institutes for Food and Drug Control (Beijing, China). Amino acids including l-methionine, l-lysine, l-phenylalanine, l-homoserine, homocysteine, l-tyrosine, l-glutamine, citrulline, l-arginine, l-cysteine, l-glutamic acid, l-alanine and L-aspartic acid were purchased from Amresco Company. Kynurenine, kynurenic acid, dopamine, 1-methyladenosine, l-xanthosine, xanthurenic acid, indole, p-cresol sulfate, uracil, p-cresol, guanosine, succinic acid, deoxyuridine, guanine, taurine and thymine were purchased from Sigma Company or Aladdin Company. Antibodies against nuclear factor kappa B p65, Nrf2, cyclooxygenase-2 (COX-2), 12-lipoxygenase (12-LP), etc. were purchased from Santa Cruz Biotechnology or Abcam Company.

Daclatasvir (DCV) and asunaprevir (ASV) were kindly provided by Bristol-Meyers Squibb (New York, NY, United States). MPA and guanosine were obtained from Sigma (Sigma-Aldrich Chemie, Zwijndrecht, the Netherlands). TAC and CSA were from Abcam (Cambridge, MA, United States). RAPA was obtained from Merck (Amsterdam, the Netherlands). Beetle luciferin potassium salt was from Promega (Promega Benelux BV, Leiden, the Netherlands). All cell lines were cultured in DMEM (Lonza Benelux, Breda, the Netherlands), with 10% fetal calf serum (Sigma-Aldrich Chemie), 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin. Huh7-ETluc cells were cultured in the presence of 500 μg/mL G418 (Life Technologies Europe BV, Bleiswijk, the Netherlands).

The templates for transcription reactions for VEGFA RNA fragments derived from pre-mRNAs were obtained in PCR using specific oligonucleotide sets (_TF/TR) having a promoter for polymerase T7. The PCR products were then purified prior to transcription reaction according to the manufacturer's protocol (A&A Biotechnology). The transcription reaction and 5′-end radiolabeling were performed as previously described with slight changes (Sznajder et al. 2016 (link)). Briefly, transcription reaction was performed in 40 µL composed of 10 µL of DNA template, 1 mM NTPs (Invitrogen), 3 mM guanosine (Sigma-Aldrich), 20 U T7 RNA Polymerase (Promega), 1× T7 transcription buffer (Promega), 40 U Rnasin Plus RNase Inhibitor (Promega). Purification of transcript was conducted on a denaturing 6% polyacrylamide gel (19:1 acrylamid:bisacrylamid) and through ethanol precipitation. For radio-labeling, 2 pmol of transcript was incubated with 2 pmol of [γ-³²P] ATP, 1 U Rnasin Plus, 10 U OptiKinase (Affymetrix), 1× reaction buffer (Affymetrix) and ddH₂0 up to 10 µL, at 37°C for 30 min. Labeled RNA was subsequently run on a denaturing 8% polyacrylamide gel (19:1) in 0.5× TBE, at 100 V for 1 h. A band of RNA was visualized on IP through FLA-5100 (FujiFilm), cut out followed by ethanol precipitation and resuspended in 20 µL ddH₂O. (CUG)₂₀ was a kind gift from Włodzimierz J. Krzyżosiak.

Lyophilized adenosine (Cas no: 58-61-7), guanosine
(Cas no: 118-00-3),
and cytidine (Cas no: 65-46-3) were obtained from Sigma-Aldrich Co.
(St. Louis, MO). Each 10 mM stock solution was prepared in 0.1 M perchloric
acid and diluted with PBS (131.5 mM NaCl, 3.25 mM KCl, 1.2 mM CaCl₂, 12.5 mM NaH₂PO₄, 1.2 mM MgCl₂, and 2.0 mM Na₂SO₄ with pH adjusted to 7.4).