Cp 91149

Neutrophils were cultured at 5 million cells/mL for 2-20 h at 37°C, in normoxia (21% O2, 5% CO2) or hypoxia (1% (3kPa) O2, 5% CO2) in two types of pre-equilibrated RPMI media 1640 with 10% fetal bovine serum (FBS) (GIBCO) and 1% Penicillin/Streptomycin, differing in the presence or absence of glucose. In the absence of glucose, dialyzed glucose-free FBS was used. To study the effects of stimulation and metabolic inhibitors, neutrophils were cultured with (100ng/mL) LPS E.coli serotype R515 (Enzo), CP-91149 (Sigma-Aldrich), 2-deoxyglucose (Sigma-Aldrich), Oligomycin A (Sigma-Aldrich) and Etomoxir (10uM) (CNIO Carlos III Therapies) (Raud et al., 2018 (link); Yao et al., 2018 (link)), Fructose-1,6-bisphosphatase-1 Inhibitor (FBP1i) (Cambridge Bioscience), MB05032 (Tocris Bioscience), 3-Mercaptopicolinic acid (HCl) (3-MPA) (Cambridge Bioscience), Glutaminase Inhibitor II, BPTES (MERCK Chemical).

Inhibitors were added to neutrophils at the following final concentrations 1 hr prior to infection, unless otherwise indicated: 20 µM WZB-117 (20 mM stock in DMSO, Sigma-Aldrich), 5 mM 2-deoxy-D-glucose (2-DG) (100 mM stock in RPMI-1640, Sigma-Aldrich), 50-100 μM 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO, 100 mM stock in DMSO, Sigma-Aldrich), 20 µM CP-91149 (20 mM stock in DMSO, Sigma-Aldrich), or 40 µg/ml chlorogenic acid (40 mg/ml stock in DMSO, Sigma-Aldrich). Q-VD-OPh (10 mM stock in DMSO, Cayman Chemical) was added to a final concentration of 10 µM, at time zero simultaneously with initiation of infection (no pretreatment).

The following reagents were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA): dimethylsulfoxide (DMSO), caffeine, CP-91149, ellagic acid, HEPES, MgCl₂, glucose 1-phosphate, and glycogen. KCl was purchased from José M. Vaz Pereira, S.A. (Sintra, Lisboa). Rabbit muscle GPa was purchased from Creative Enzymes^® (Shirley, NY, USA). The reagent for colorimetric phosphate quantitation, BIOMOL^® Green, was acquired from Enzo Life Sciences, Inc. (Miraflores, Portugal).