S nitrosoglutathione gsno

Human (primary) hepatocytes were obtained from the National Institutes of Health (NIH) – funded Liver Tissue and Cell Distribution System core at the University of Pittsburgh. The hepatocytes were cultured in Hepatocyte Maintenance Medium (LONZA, Walkersville, MI) with 5% newborn calf serum. The mouse hepatocytes were isolated from normal mice by an in situ collagenase (type IV) (Sigma Aldrich, Natick, MA) perfusion technique, modified as described previously (Tsung et al. 2006 (link)). Unless indicated, cells were stimulated with 250 U/mL human or mouse IFNγ (R&D Systems, or Roche Pharmaceuticals). L-NIL (N⁶-(1-Iminoethyl)-L-lysine hydrochloride) (Cayman Chemical, Ann Arbor, MI) and BYK191023 (2-[2-(4-methoxypyridin-2-yl)-ethyl]-3H-imidazo [4,5-b]pyridine) (Santa Cruz Biotechnology, Santa Cruz, CA), Romidepsin (also known as Istodax) (MedChemExpress, Monmouth Junction, NJ), GSNO (S-Nitrosoglutathione) (Sigma Aldrich, Natick, MA), SNAP (S-Nitroso-N-Acetyl-D,L-Penicillamine) (Cayman Chemical, Ann Arbor, MI) were performed according to the manufacture’s protocol.

Human Alpha Thrombin (HT 1002a) and human plasminogen (HPg 2001) were acquired from Enzyme Research Laboratories (Bulimba, Australia). Human Interleukin-1β was purchased from Invitrogen Thermo Fisher Scientific Inc (Victoria, Australia). Recombinant human tissue-type plasminogen activator (t-PA) and d-dimer (D2D) ELISA Kit were purchased from Antibodies-Online Inc. (Atlanta, USA). S-nitrosoglutathione (GSNO) was ordered from Sigma Aldrich (Sydney, NSW, Australia).

Determination of SNO content was performed according to the modified method of Rustérucci et al. (2007) (link). Leaf samples (400 mg) were homogenized in a mortar with liquid nitrogen, then with 2 ml 100 mM Tris-HCl buffer, pH 6.8, and centrifuged (15,000 × g, 15 min). The extracts were incubated for 5 min separately with an equivalent volume of solution A (1% sulfanilamide dissolved in 0.5 M HCl) or of solution B (solution A plus 0.2% HgCl₂) which by hydrolyzing SNO allow to form diazonium salt. After 5 min, an equivalent volume of solution C [0.02% N-(1-naphthyl)ethylenediamine dihydrochloride dissolved in 0.5 M HCl] was added both to samples with A and B reagents. Absorbance related to formation of the azo dye product, obtained through reaction of diazonium salts with reagent C, was read at 550 nm after 5 min incubation. SNO were quantified on the basis of the difference of absorbance between samples with solution B and appropriate blank solution (A) (B − A), comparing the values with a standard curve made from the solution of S-nitrosoglutathione (GSNO) (Sigma-Aldrich). The results were expressed in nmol SNO per mg of protein.

The GBM cell lines (T98G, U87MG, A172, U251, LN18) used in this study were grown in DMEM, (Euroclone; Milan, Italy) with 10% fetal bovine serum (FBS) (Euroclone). T98G and U87MG cell proliferation was tested using ³H-thymidine incorporation assay [56 (link)] and cell viability was tested by MTS colorimetric assay (Promega, Milan, Italy). Glioblastoma stem cell line 83 was derived from patient derived glioblastoma obtained after informed consent and surgery, validated and grown in stem cell medium as previously reported [57 (link)]. The MAPK inhibitors PD98059 and U0126 were from Calbiochem (Milan, Italy). hEGF and hbFGF were from Immunological Sciences (Rome, Italy). Olaparib was obtained from Selleckchem (Milan, Italy). S-nitrosoglutathione (GSNO, a synthetic nitric oxide donor) and type C natriuretic peptide (CNP) were from Sigma-Aldrich (Milan, Italy).