Menadione sodium bisulfite

The metabolic activity of microbial biofilms was calculated using a colorimetric assay. Biofilm formation was performed, by using a 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carbox-anilide (XTT) (Sigma, St. Louis, MO, USA) reduction assay as an indicator of cell viability and proliferation which was prepared in Ringers lactate (0.5 mg mL⁻¹). The solution was filter-sterilized (0.22 μm pore size) and then stored at −70°C. Prior to each assay, XTT stock solution was mixed with menadione sodium bisulfite (10 mM, Sigma Chemical Co., St. Louis, USA). After 48 h of incubation, the tissue conditioner disks were transferred to a new tissue culture plate and washed twice with sterile PBS. In the following, 500 μL aliquot of XTT/menadione was added to each well of 24-well plates. The plates were incubated at 37°C in a dark room (3 h). Finally, the colorimetric changes were measured at 570 nm by using a microplate reader (BMG Labtech, Berlin, Germany). The cell viability percent was calculated as follows: cell viability percent = (absorbance of test well/absorbance of control well) × 100.

U2OS cells were grown at 37 °C in a 5% CO₂ atmosphere in DMEM (Gibco) growth medium supplemented with 10% of fetal bovine serum (FBS; Gibco) and 100 U/mL penicillin-streptomycin (Gibco). In order to induce DNA oxidative damage, cells at about 80% confluency were challenged with menadione sodium bisulfite (Sigma-Aldrich, St. Louis, USA, CAS 130-37-0) in serum-free DMEM for the indicated periods of time. To inhibit OGG1, 10 μM TH5487 or 0.1% DMSO (VWR chemicals, Radnor, USA) were used according to the indicated experimental scheme for the indicated time periods.

Phenotype microarray plates and standard components for yeast phenotypic analysis were obtained from Biolog Inc. (Hayward, CA). Wild type R. toruloides IFO0880 was precultured to log phase in LB broth at 30°C, 200 RPM in 10 mL culture tubes. Cells were centrifuged 5 min at 3000 RCF, 22°C, washed twice in sterile water, then resuspended OD 600 of 0.005 in Biolog inoculation fluid IFY-0 with 1 μM nicotinic acid (Sigma, N4126), 1 μM myo-inositol (Sigma, I5125), 1 μM thiamine HCl (Sigma, T1270), 1 μM p-aminobenzoic acid (Sigma, A9878), and 1 μM calcium pantothenate (Sigma, 21210) plus Biolog dye mix E (a proprietary, tetrazolium-based dye) and 1 μM menadione sodium bisulfite (Sigma, M5750). For nitrogen, phosphorous, and sulfur sources 100 mM glucose was added to the inoculation fluid. Hundred microliters of the cell suspension was added to each well in plates PM1, PM2, PM3, and PM4. Plates were sealed with clear sealing film (Axygen, CTP-103) and incubated for 120 h at 30°C in the dark. Respiration in each condition was detected by measuring reduction of the dye by comparing absorbance at 590 nm to absorbance at 750 nm.

For experiments using low glucose in C2C12 or HK-2 cells, standard high-glucose media was replaced with low-glucose DMEM (5 mM) or low-glucose DMEM/F12, and cells were incubated for 24 h before harvesting polar metabolites. To characterize erythritol synthesis during oxidative stress, cells were exposed to menadione sodium bisulfite (Sigma) or hydrogen peroxide (H₂O₂) (Thermo Scientific) at the indicated dose for 2 h, after which polar metabolites were extracted. Cells were incubated with the aldose reductase inhibitor Sorbinil (Sigma) at the indicated dose for 24 h before harvesting polar metabolites.