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7 protocols using sodium succinate dibasic hexahydrate

1

Helminth infection and succinate effects

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8–12-wk-old female C57BL/6 mice bred in-house or purchased from Envigo UK housed in individually ventilated cages were used throughout this study. All animal studies were performed under UK Home Office Licence and approved by the University of Glasgow Ethical Review Board.
Infections employed H. polygyrus and N. brasiliensis, both maintained as previously described (Camberis et al., 2003 (link)). HpES was collected as previously described (Johnston et al., 2015 (link)). For succinate experiments, sodium succinate dibasic hexahydrate (Sigma-Aldrich) was dissolved in autoclaved tap water to 100 mM, filter-sterilized, and given to mice as their drinking water. To administer HpES in vivo, 5 µg in 100 µl PBS was given i.p. daily from day −1 to day +4 relative to addition of succinate to drinking water.
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2

High-Resolution Respirometry SUIT Protocol

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For high-resolution respirometry SUIT protocols, following chemicals (catalog number) were used: adenosine 5′-diphosphate sodium salt (A2754), antimycin A from Streptomyces sp. (A8674), L-glutamic acid monosodium salt hydrate (G1626), L(−)malic acid sodium salt (M1125), sodium pyruvate (P2256), sodium succinate dibasic hexahydrate (S2378), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (C2920) and rotenone (R8875) from Sigma-Aldrich, cytochrome c (24,804) from Merck, rac-glycerol 1-phosphate disodium salt hexahydrate (sc-215,789) from Santa Cruz Biotechnology, and ( ± )-octanoylcarnitine chloride (15,048) from Cayman.
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3

A. baumannii Growth in Minimal Media

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Overnight cultures of A. baumannii were subcultured 1:50 in LB for 1 h. Cultures were then inoculated 1:100 into M9 minimal media supplemented with Vishniac’s trace metal mix with or without ZnCl2 as previously described (Nairn, et al., 2016 (link)). M9 minimal media supplemented with full Vishniac’s trace metal mix contained 36 μM Fe, 5.2 μM Mn, 3.2 μM Cu, 1.8 μM Mo, 13.4 μM Co and 27.2 μM Zn. Sodium succinate dibasic hexahydrate (Sigma) or sodium fumarate dibasic (Sigma) were used as the sole carbon source at 0.5 % (w/v). Riboflavin (Sigma) was supplemented with a final concentration of 10 μg/mL, and OD600 was monitored over time. Data are representative of at least 3 independent experiments.
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4

Cytotoxicity Evaluation of DOX in DMEM

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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.
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5

Metabolite supplementation experiments

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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.
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6

Hypoxia and Succinate Effects on hPDLCs

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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.
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7

Modulating Tumor Cell Responses

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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)
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