Sodium pyruvate

HT (purity ≥98%) was obtained from Extrasynthese, France. Dulbecco’s modified Eagle’s medium (DMEM) and sodium pyruvate were from Capricorn Scientific, Germany, foetal bovine serum (FBS) was from Sigma, USA, and Trypan Blue Solution, 0.4% from Thermo Fisher, USA. Primary antibodies mTOR (2972 S), p-mTOR (2971 S), S6 (2217 S) and p-S6 (2211 S) were purchased from Cell signaling Technology, USA; HIF-1α (A300–286A) from Bethyl, USA; PARP-1 (C-2-10) from Calbiochem, Germany; anti-poly(ADP-ribose) (α-PAR) (4355-MC) from Trevigen, USA; α-Tubulin antibody (T5168) from Sigma, USA and FIH-1 (sc-26219) from Santa Cruz, USA. Apoptosis was quantified using the FITC Annexin V Apoptosis Detection Kit with PI (ANXVKF, Immunostep, Spain). RNA was isolated using the RNeasyPlus Mini kit (Qiagen, Germany). cDNA Synthesis Kit for RT-qPCR and iTaq UniverSYBR for Real-time PCR were from Bio-Rad, USA. Primers were synthesized by Biomedal S.L. (Spain). ARNT siRNA (s1613 and s1615), scramble siRNA (sc-37007) and the transfection reagent jetPRIME were from Ambion, Santa Cruz and Polyplus Transfection, USA, respectively. HIF-1α siRNA was from Sigma (forward 5′-CUGAUGACCAGCAACUUGA-3′, reverse 5′-UCAAGUUGCUGGUCAUCAG-3′).

The human glioblastoma cell line U251 was obtained from the European Collection of Animal Cell Cultures (Salisbury, UK). The cells were incubated at 37°C, in a humidified atmosphere with 5% CO₂, in the RPMI cell culture medium (Gibco, Life Technologies, MA, USA) supplemented with 5% fetal calf serum (FCS), 2 mM L-glutamine, 10 mM sodium pyruvate, and penicillin/streptomycin (all from Capricorn Scientific, Ebsdorfergrund, Germany). Cells were prepared for experiments using the conventional trypsinization procedure with trypsin/EDTA and seeded in 96-well flat-bottom plates (1 × 10⁴ cells/well) for the $\underset{¯}{\text{cell\hspace{0.17em}viability}}$ assessment, 6-well plates (2.5 × 10⁵ cells/well) for flow cytometric analysis, or 60 mm Petri dishes (1 × 10⁶ cells) for the electron microscopy, immunoblot, and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR).