3 bromopyruvate

Polar metabolite standards, stable isotope standards [isoleucine (¹³C₆, ¹⁵N), alanine (2,3-¹³C₂), aspartic acid (U-¹³C₄, ¹⁵N), glutamine (U-¹³C₅, U-¹⁵N₂), uracil (1,3-¹⁵N₂), palmitoyl–CoA (¹³C₁₆), AMP (¹³C₁₀, ¹⁵N₅), malic acid (¹³C₄), succinic acid (1,4-¹³C₂), and pyruvate (3-¹³C)] were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Ammonium acetate (LC-MS grade) and 3-bromopyruvate (>99%) were purchased from Sigma (St. Louis, MO, USA). HPLC grade solvents were purchased from Fisher Scientific (Hampton, NH, USA).

For inhibitor studies, 24 h after plating inhibitor was added at the indicated times. The cells were counted with a TC20 cell counter (BioRad, Hercules, CA, USA) using Trypan blue for the detection of viable cells. The cell pellet was weighed, snap frozen in liquid nitrogen and stored until analysis at −20 °C. The glycolysis/glutaminolysis inhibitor 3-bromopyruvate (Sigma-Aldrich, St. Louis, MO, USA) dissolved in phosphate buffered saline (PBS) was added from a sterile stock solution to a final concentration of 0.25 mM.

For inhibitor studies the following reagent were used: The glycolysis inhibitors 3-bromopyruvate (Sigma-Aldrich, St. Louis, MO, USA) and L-glyceraldehyde (Sigma-Aldrich, St. Louis, MO, USA) solved in PBS were added from a sterile stock solution to final concentration of 0.25 mM and 1 mM, respectively. SR13800 (Tocris, Bristol, United Kingdom) and AZD3965 (Selleck Chemicals, Houston, TX, USA) - inhibitors of MCT1 -solved in DMSO were added from a sterile stock solution to final concentration of 0.1 µM and 10 µM, respectively. AKT inhibitor MK-2206 (Selleck Chemicals, Houston, TX, USA) solved in DMSO were added from a sterile stock solution to final concentration of 1 µM. mTOR inhibitors Rapamycin (Calbiochem, Billerica, MA, USA) and OSI027 (Selleck Chemicals, Houston, TX, USA) solved in DMSO were added from a sterile stock solution to final concentration of 10 µM. AMPK inhibitor Dorsomorphin and AMPK activator AICAR (both Selleck Chemicals, Houston, TX, USA) solved in DMSO were added from a sterile stock solution to final concentration of 1 µM or 1 mM, respectively. As control, inhibitor solvent was used.

3-Bromopyruvate was obtained from Sigma-Aldrich (St. Louis, MO, USA). A fresh stock solution of 20 mM 3BrPA in PBS was prepared for each experiment.

DPI and 3-bromopyruvate were purchased from Sigma-Aldrich (St. Louis, MO). CM-H2DCF-DA, DAF-FM, HEt, and 2-NBDG were purchased from Invitrogen/Molecular Probes (Carlsbad, CA). SP600125 was acquired from EMD Biosciences (Calbiochem, San Diego, CA). DPI was dissolved in dimethyl sulfoxide (DMSO) and freshly diluted in culture media before used. The final DMSO concentration was less than 0.1% (v/v). In addition, 3-bromopyruvate was dissolved in water and neutralized with NaOH immediately before use in cell culture. The rabbit polyclonal anti-p22^phox antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-Akt and anti-phospho-Akt (Ser473) antibodies as well as rabbit monoclonal anti-c-Jun and anti-phospho-c-Jun (Ser63) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Determination of interactions between the selected plant extracts and substances with documented antimicrobial activity against H. pylori 8064 (amoxicillin (AMX), 3-bromopyruvate (3-BP), or sertraline (SER)) [22 (link),34 (link),35 (link)], all from Sigma-Aldrich (St. Louis, MO, USA) was performed using the checkerboard assay [34 (link),35 (link)]. To obtain this, four 12-well plates were joined together to form a 48-well panel. Each well of the titration plates was filled with 1 mL of BHI with 5% fetal calf serum, bacteria with a density of 10⁷ CFU/mL, and a mixture of both tested components (one of the extracts (corresponding to MIC–1/64 × MIC) and one of the aforementioned synthetic substance (corresponding to MIC–1/16 × MIC)). Such titration plates were then directed to a 3-day microaerophilic culture at 37 °C and 100 rpm (0.1× g) shaking. The interactions between the tested components were determined by calculating the fractional inhibitory concentration index (FICI), for which the values ≤0.5, 0.5–1, and >1 were considered as synergistic, additive, and neutral, respectively [33 (link)].