Sodium succinate dibasic hexahydrate

Dulbecco’s Modified Eagle Medium (DMEM) powder was purchased from Gibco by Life Technology (Thermo Fisher Scientific, Waltham, MA, USA). Calcium chloride dihydrate (CaCl₂·2H₂O), sodium bicarbonate (NaHCO₃), sodium hydrogen phosphate (Na₂HPO₄), potassium dihydrogen phosphate (KH₂PO₄), sodium citrate tribasic dihydrate, sodium succinatedibasic hexahydrate and trypsin-ethylene diaminetetraacetate (EDTA) salts were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulphoxide (DMSO), thiazolyl blue tetrazolium bromide (MTT), nonidet P40 substitute (NP40) and anti-cancer drug DOX were acquired from Sigma-Aldrich (St. Louis, MO, USA). DMEM liquid media, foetal bovine serum (FBS), TrypLE Express, and penicillin-streptomycin were procured from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride and potassium chloride salts were bought from Fischer Scientific (Loughborough, UK). Acetonitrile (ACN), hydrochloric acid (HCl), methanol and glycerol were from Fischer Scientific (Loughborough, UK). DOX was dissolved in distilled water and 8.62 mM stock solution was prepared.

In certain experiments (one of) the following compounds was/were added to the medium: sodium pyruvate (#11360070, Life Technologies, Carlsbad, CA, USA), l-aspartic acid (#A7219, Sigma-Aldrich), 2-ketobutyric acid (#K401, Sigma-Aldrich), oxaloacetic acid (#O4126, Sigma-Aldrich), sodium l-lactate (#L7022, Sigma-Aldrich), l-(-)-malic acid (#M1000, Sigma-Aldrich), sodium succinate dibasic hexahydrate (#S9637, Sigma-Aldrich), dimethyl α-ketoglutarate (#349631, Sigma-Aldrich), uridine (#U3003, Sigma-Aldrich), β-nicotinamide adenine dinucleotide hydrate (#43410, Sigma-Aldrich), Oleamide (#O2136, Sigma-Aldrich) These compounds were dissolved directly in the medium after which its pH was adjusted to 7.2 with NaOH. Media were freshly prepared prior to each experiment.

1 × 10⁶ cells were plated in 10 cm. Cells were cultured in either the normal oxygen condition or the hypoxia condition (1% oxygen) in the hypoxic chamber (Thermo Scientific, USA). If oxygen tension rose above the desired level, nitrogen gas was automatically injected into the system to replace the excess oxygen. For the succinate supplement group, sodium succinate dibasic hexahydrate (Sigma-Aldrich, USA) was added to the culture medium at the designated concentration (1, 5, or 25 mM). To inhibit HIF-1α activity, hPDLCs were pretreated with the HIF-1α-specific inhibitor BAY 87-2243 (5 nM; Selleck, USA) for 3 h.

Inhibitors and activators were added 2 h prior to the treatment with the antitumoral agents (24 h). 3PO (Sigma, SML-1343; 10 µM), TOFA (Sigma, T6575; 1 µg/mL), C968 (Calbiochem, 352010; 40 nM, Madrid, Spain), sodium lactate (Sigma, L7022), sodium succinate dibasic hexahydrate (Sigma, S9637), di-methyl-fumarate (Sigma, 242926); lactacystine (Sigma, L6785; 10 µM), BBG (BioRad 161-0400; 10 µM, Alcobendas, Spain), LY294002 (Calbiochem, 440202; 10 µM), NFκB inhibitor SN50 (Calbiochem, 481480; 18 µM), actinomycin D (GE HealthCare Biosciences, 116128; 3 µg/mL), insulin (Sigma, 91077C; 100 nM), IFNγ (PeproTech, 300-02; 20 ng/mL), dexamethasone (Sigma, D4902; 1 µg/mL), rapamycin (Sigma, R0395, 10 nM), cycloheximide (Sigma, C6255; 10 µg/mL), rosiglitazone (Calbiochem, CAS155141-29-0; 1 µM), IFNα (Sigma, IF007, 20 ng/mL), MCC950 (Sigma, PZ0280; 300 nM), GW3965 (Merck, G6295, 1 μM), RAF inhibitor (Sigma, 475958; 10 μM), and PD98059 (Merck, HY-12028; 10 μM)