Stable glutamine

The SH-SY5Y human neuroblastoma cells were purchased from ECACC (ref 94030304). They were cultured in high-glucose D-MEM with stable glutamine (PAA Laboratories) supplemented with 10% dialysed FCS (PAA Laboratories) and a solution of stabilised Antibiotic-Antimycotic (Sigma-Aldrich). SH-SY5Y cells were plated in 6-well plates at a density of 7.10⁵ cells/well. The next day, culture medium was removed and cells were incubated for 48 h with increasing concentrations of recombinant hIFN-α (1, 10, 100, 1000 and 10,000 IU/ml, n = 8 for each concentration, PBL Biomedical Laboratories). Untreated cells were taken as the control group (vehicle, n = 8). After incubation, cells were lysed in 600 μl of RLT lysis buffer (Qiagen, RNeasy Plus Mini Kit). RNA isolation and purification were carried out as described by the manufacturer (Qiagen, RNeasy Plus Mini Kit). For homogenisation, cell lysates were passed through QIAshredder spin columns (Qiagen) and the flow-throughs were then transferred to gDNA eliminator spin columns. RNAs were purified on RNeasy spin columns and eluted with 40 μl of RNase-free water. Total RNAs were quantified with the Quant-IT RNA BR assay-500 and a Qubit Fluorometer (Invitrogen). The quality of RNAs (500 ng) was checked on a 1.5% agarose gel.

NIH/3T3 fibroblast (ATCC-CRL-1658) and HeLa (ATCC-CCL-2) cells were grown in T25-flasks as a monolayer in DMEM with high glucose (4.5 g l⁻¹) and stable glutamine (PAA Laboratories GmbH, Austria). The medium was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (PAA Laboratories GmbH), referred to as cell culture medium. Exponentially growing cultures were maintained in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C, and in these conditions, the plating efficiency was 70–90% and the cell duplication time was 12–14 h. Cell cultures were passaged twice weekly using trypsin–EDTA (PAA Laboratories GmbH) to detach the cells from their culture flasks and wells. The cells were pelleted by centrifugation at 260 g for 5 min. The supernatant was removed and cell pellets were re-suspended in assay medium and counted using a Malassez counting chamber.