β nicotinamide adenine dinucleotide

NAD⁺ and NADH concentrations were determined using the bioluminescent NAD/NADH-Glo Assay from Promega (Madison, WI). Cells were plated at a density of 1.5–3 × 10⁴ cells per well in 250 μL complete media on 96-well plates. For determining the effects of treatments on NAD⁺/NADH ratios, cells were left untreated or drugs added, and all cells were incubated for times specified in figures. Nanomolar concentrations of NAD⁺ and NADH were determined following manufacturer’s instructions by comparison to a standard curve consisting of dilutions of β-Nicotinamide adenine dinucleotide (N8285, Sigma).

According to Ma et al.^{22 (link)}, the β-Nicotinamide adenine dinucleotide (NADH, Sigma-Aldrich) was prepared fresh each time as a stock solution of 428 µM in a 0.2 mM Tris-HCl, pH 7.5 and stored at 4 °C. The absolute concentration of the free NADH stock solution for calibration was determined prior to use in a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific Inc) at 340 nm. The actual free NADH concentration was calculated based on the Beer-Lambert law $$\mathit{A}=\mathit{\epsilon }\mathit{bc}$$ where A is the readout absorbance; ε is the extinction coefficient of NADH (6.22 at 340 nm); b is the length of light path (1 mm) and c refers to concentration of NADH in the solution.

5‐Methyl‐10,11‐di‐hydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate (MK801) and isolectin‐B4‐FITC were purchased from Tocris (Bristol, United Kingdom). 7‐aminoactinomycin D (7‐AAD), lactate dehydrogenase (LDH), phenylmethanesulfonyl fluoride (PMSF), phosphatase inhibitor, protease inhibitor cocktails 2 and 3, Hoechst 33258, glutamate, β‐nicotinamide adenine dinucleotide (NADH), sodium pyruvate, β‐nicotinamide adenine dinucleotide 2′‐phosphate reduced tetrasodium salt hydrate (NADPH), MgSO₄, and nitro blue tetrazolium were from Sigma Aldrich (Saint‐Quentin Fallavier, France). The ratiometric intracellular calcium probe Fura‐2, pluronic^® F‐127 and Cell Tracker green were from Invitrogen (Cergy Pontoise, France). The Apo‐ONE homogeneous caspase‐3/7 kit was provided by Charbonnières‐les‐Bains, France. Isoflurane was from CSF (Cournon, France). Characteristics of the antibodies used in the present study for immunohistofluorescence and Western blot experiments are summarized in Table S1.

Snap-frozen FSEs from -80°C were thawed and fixated in 1% paraformaldehyde (Sigma) for 2 h at 4°C. The FSEs were then put in 20% sucrose (Sigma) in PBS solution overnight at 4°C. FSEs were embedded in Tissue Tek OCT (Sakura Finetek Europe B.V., Alphen aan de Rijn, Netherlands) and sections of 10 µm were cut using a cryotome (Slee MNT, Adamas Instruments B.V., Rhenen, Netherlands). Dried sections were washed in PBS and incubated with LDH solution (2 mM Gly-Gly (Sigma); 0.75% NaCl (Sigma); 5% polypep (Sigma); 1.75 mg/ml β-nicotinamide adenine dinucleotide (Sigma); 3 mg/ml nitroblue tetrazolium (Sigma) in demineralized water of pH 8) for 3 h at 37°C. Sections were washed in tap water at 50°C and in PBS and then stained with Eosin Y for 4 min. Sections were then put in PBS for 1 sec, acetone for 30 sec, acetone/xylene (1:1) for 1 min and xylene for 1 min, before embedding with Eukit Mounting Medium.

Pd (99.95%) was obtained from Silver Gold Bull. β-Nicotinamide adenine dinucleotide hydrate (≥99%), β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (≥97%, HPLC), propionic aldehyde (reagent grade, 97%), ammonium chloride, bovine serum albumin, L-lactic dehydrogenase from rabbit muscle, alcohol dehydrogenase from Saccharomyces cerevisiae, L-alanine dehydrogenase from Bacillus subtilis, sulfuric acid (95.0–98.0%), H₂O₂ solution (30 wt.% in H₂O) and dimethylsulfone (quantitative NMR standard, TraceCERT) were purchased from Sigma Aldrich and used as received. Sodium pyruvate (≥99%) was purchased from Fisher Scientific. 68–70% Nitric acid was obtained from VWR. Pt gauze (52 mesh, 99.9%) and Pt wire (0.5 mm, 99.95%) were obtained from Alfa Aesar. Ag/AgCl reference electrodes (RE5B) were purchased from BASi.

Neisseria gonorrhoeae was grown overnight at 37°C and 5% CO₂ on agar plates containing gonococcal base (GC) agar [10 g/l Bacto agar (BD Biosciences, Bedford, MA, United States), 5 g/l NaCl (Roth, Darmstadt, Germany), 4 g/l K₂HPO₄ (Roth), 1 g/l KH₂PO₄ (Roth), 15 g/l Proteose Peptone No. 3 (BD), 0.5 g/l soluble starch (Sigma-Aldrich, St. Louis, MO, United States)] supplemented with IsoVitaleX (IVX): 1 g/l D-Glucose (Roth), 0.1 g/l L-glutamine (Roth), 0.289 g/l L-cysteine-HCL × H₂0 (Roth), 1 mg/l thiamine pyrophosphate (Sigma-Aldrich), 0.2 mg/l Fe(NO₃)₃ (Sigma-Aldrich), 0.03 mg/l thiamine HCl (Roth), 0.13 mg/l 4-aminobenzoic acid (Sigma-Aldrich), 2.5 mg/l β-nicotinamide adenine dinucleotide (Roth) and 0.1 mg/l vitamin B₁₂ (Sigma-Aldrich). GC medium is identical to the base agar composition but lacks agar and starch.
E. coli was grown in LB (Lysogeny Broth, Roth) medium or on LB agar plates (15 g/l Bacto agar (BD Biosciences, Bedford, MA, United States) at 37°C.
For N. gonorrhoeae antibiotics were used at the following concentrations: 2.5–5 μg/ml erythromycin (Thermo-Fisher), 100 μg/ml streptomycin (Sigma-Aldrich), 10 μg/ml chloramphenicol (Sigma-Aldrich). For E. coli antibiotics were used at the following concentrations: 50 μg/mL kanamycin (Roth).

The following materials were used in this study: sulfo-LC-SPDP (sulfosuccinimidyl 6-(3′-(2-pyridyldithio)propionamido)hexanoate; ThermoScientific, Waltham, MA, USA, 21650), TCEP-HC (Tris(2-carboxyethyl)phosphine hydrochloride Thermo Scientific, Waltham, MA, USA, 20490), EDTA (ethylenediaminetetraacetic acid; Sigma Aldrich, Burlington, MA, USA, E9884), MicroDeck^TM-G-150 surfaces (Bioactive Surfaces, Galapagar, Madrid, Spain), lactate dehydrogenase (LDH; Sigma Aldrich, Burlington, MA, USA, L1006-12.5KU), biotinylated anti-lactate dehydrogenase (anti-LDH) antibodies (IgG polyclonal antibody; Origene, Rockville, MD, USA, AP21329BT-N), poly(ethylene glycol)-(N-hydroxysuccinimide 5-pentanoate) ether 2-(biotinylamino)ethane (NHS–PEG–biotin; Sigma Aldrich, Burlington, MA, USA, 757799-100μγ), streptavidin (Sigma Aldrich, Burlington, MA, USA, S4762-5 mg), DeepTip^TM SiN R11 atomic-force microscopy probes (Bioactive Surfaces, Galapagar, Madrid, Spain), Goat Anti-Rabbit IgG H&L Alexa Fluor^® 488 (Abcam, Cambridge, UK, ab150077), 5-iodoacetamidofluorescein (5-IAF; Thermo Scientific, Waltham, MA, USA, 62246), sodium pyruvate (P2256 Sigma-Aldrich, Burlington, MA, USA,), β-nicotinamide adenine dinucleotide (NADH; N8129-500 mg), and glutaraldehyde (50 wt. % in H₂O, Sigma Aldrich, Burlington, MA, USA, 340855).