La assay kit

S. cerevisiae strain BY4741 was cultivated in liquid yeast extract peptone dextrose (YPD) medium containing 20 g/L glucose, 10 g/L yeast extract, and 20 g/L peptone. The recombinant strains with the Ura3 gene were cultivated in liquid SC-Ura medium (synthetic complete medium without uracil; 6.7 g/L yeast nitrogen base without amino acids, 20 g/L glucose, 0.1 g/L leucine, 0.02 g/L histidine, and 0.02 g/L tryptophan). The recombinant strains without the Ura3 gene were cultivated in liquid SC-5-FoA medium (synthetic complete medium with 5-FoA; 1 g/L 5-FoA, 6.7 g/L yeast nitrogen base without amino acids, 20 g/L glucose, 0.1 g/L leucine, 0.02 g/L histidine, 0.02 g/L tryptophan, and 0.5 g/L uracil). The growth of the yeast strains during cultivation were measured at a wavelength of optical density (OD) 600 nm using a spectrophotometer. The lactic acid concentrations were measured using the LA Assay Kit (BC2235, Solarbio).

Quercetin and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). The mouse macrophage-like cell line, RAW264.7 (CSTR:19375.09.3101MOUTCM13), was acquired from the National Collection of Authenticated Cell Cultures, the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s modified eagle’s medium (DMEM) high glucose were purchased from Gibcol Life Technology (Thermo Fisher, Waltham, MA, USA). An enhanced ATP Assay Kit was acquired from Beyotime^® Biotechnology (Shanghai, China). anti-PGC1 (Cat# ab191838), anti-glutathione peroxidase 4 (Cat# ab125066), anti-AMPK alpha 1 (Cat# ab32047) and anti-SIRT1 (Cat# ab110304) antibodies were purchased from Abcam Biotechnology (Cambridge, MA, USA). Phospho-AMPK (Thr172) (Cat# 2535s) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The 3-4,5-dimethylthiazole-z-yl-3,5-diphenyltetrazolium bromide (MTT), trypsin, dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), LA Assay Kit, Micro Pyruvate (PA) Assay Kit and Mitochondrial Membrane Potential Assay Kit with JC-1 were purchased from Solarbio Science & Technology Co. Ltd. (Beijing, China), and stored at −20 °C. EX-527 and compound C were obtained from Med Chem Express (Monmouth Junction, NJ, USA).

All cell lines were inoculated in 25 cm^{2 (link)} culture bottle containing 6 mL complete medium (6 replicates) with an initial cell count of 10⁵. For the next 5 days, the cell cultures were removed from flasks twice a day and transferred to 15 mL sterile centrifuge tubes. After centrifugation at 200 r.c.f. for 5 min, the culture medium supernatant was cryopreserved at −80 °C. Then biosensor strains were inoculated into 24-well plates containing 800 μL LB medium (supplemented with corresponding antibiotics). After that, 200 μL of stored cell cultures supernatant was thawed and incubated with the above strains. pH test paper was used to detect the pH of cell cultures supernatant. Lactate concentration of cell cultures supernatant was measured using LA Assay Kit (BC2235, Solarbio). The hypoxic biosensor was tested in a constant temperature incubator under hypoxic or normoxic condition. All bacteria were cultured for 12-16 hours, and the fluorescence and absorbance of the biosensor strains were determined using a microplate reader (Thermo Fisher Scientific, USA).

The biofilms for lactic acid measurement were incubated in 24-well plates with 1 ml of pH 5 BHI broth (Zhang et al., 2015 (link)). Follow the instructions (LA Assay Kit, Solarbio, China) to monitor the lactic acid production at OD_{340 nm}. The supernatant of biofilm incubated in pH 5 BHI broth for 2, 4, 6, 8, and 16 h was used for pH measurement by Starter (Starter 3100, United States).
Furthermore, to evaluate the effect of bedaquiline on the lactic acid production of mature S. mutans biofilm (obtained by culturing in pH 7 BHI for 16 h), the shock assay was designed and performed. Based on the CFU count results of the antibacterial effect of bedaquiline with time gradient, the mature S. mutans biofilm was shocked for 2 h in pH 5 BHI with the incorporation of 2.5, 4, and 10 mg/L of bedaquiline, and then the lactic acid production of the shocked biofilms was detected as above. In parallel, pH changes of the shocked biofilms were recorded.

Cells were seeded into six-well plates at a density of 1 × 10⁶ cells in 2 ml RPMI 1640 medium per well and cultured overnight. Cells were then treated with or without 10 μM KU60019 and maintained at 1% O₂ for 10–12 h. Glucose consumption, lactate production, ATP production, citrate production, succinate production, fumarate production, and PFKP and CS activity were detected using glucose assay kit (Solarbio® BC2500), LA assay kit (Solarbio® BC2230), ATP content assay kit (Solarbio® BC0300), citric acid (CA) content assay (Solarbio® BC2150), micro mitochondrial citric acid content assay kit (Solarbio® BC2175), succinate colorimetric assay kit (Sigma® MAK184), pyruvate (PA) assay kit (Solarbio® BC2200), acetyl-CoA assay kit (Solarbio® BC0980), fumarate assay kit (Sigma® MAK060), PFKP test kit (Nanjing Jiancheng Bioengineering Institute A129), and citroyl synthetase kit (Nanjing Jiancheng Bioengineering Institute A108) according to instruction of the manufacturers, respectively. All experiments were performed at least three times and the data were normalized by the cell numbers or protein content.