Phenazine methosulphate

The reaction mixture was prepared in such a way that phenazine methosulphate (Sigma Aldrich, USA) (186 μM) and sodium phosphate buffer (Sigma Aldrich, USA) (0.052 mM) were mixed in a volume of 50 μL and 600 μL respectively. The renal supernatant was prepared by centrifuging the homogenate at 1500 g for 10 min, and then further for 10,000 g for 15 min. This supernatant was added to the previously prepared reaction mixture followed by the addition of 100 μL NADH (780 μM). After 1 min, the reaction was stopped by the addition of 500 μL glacial acetic acid (Sigma Aldrich, USA). The optical density of chromogen was quantitatively estimated at 560 nm on a spectrophotometer, and enzyme activity was calculated such that the enzyme concentration required for inhibiting 50% chromogen in 1 min [29 ].

Ethanol, methanol, ethyl acetate, n-hexane, polyvinylidene fluoride (PVDF) membrane, phosphate-buffered saline tablets, RNAwait solution, tissue protein extraction (T-PER) kit, protein assay dye, sample buffer (2X Laemmli), trizma base, acrylamide, bis-acrylamide, sodium dodecyl sulfate (SDS), ammonium persulfate (APS), tetramethylethylenediamine (TEMED), glycine, skim milk, KCl, NaCl, Tween 20 and H₂O₂, guaiacol, phenazine methosulphate, glacial acetic acid, sulfosalicylic acid, DTNB ascorbic acid, trichloroacetic acid, and thiobarbituric acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All the chemicals and reagents were stored at the required temperature according to the material safety and data sheet for experimental purposes.

The inhibiting effects on C. albicans biofilms were assessed as described previously by^{71 (link)}. Briefly, 8 × 10⁴ C. albicans cells were added to the wells of a 96 well plate, together with the samples (supernatant, lactic acid, Msp1) or controls (MRS or H₂O). After incubation for 24 h at 37 °C, the biofilms were washed twice and then 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (90 µl, 1 mg/ml) (Sigma Aldrich) and phenazine methosulphate (10 µl, 0.2 mg/ml) (Sigma Aldrich) were added to the wells. After a second incubation (37 °C, 30 minutes, in the dark), the absorbance at 492 nm was measured using a Synergy HTX multi-mode reader (Biotek, Drogenbos, Belgium).

CCl₄, reduced glutathione (GSH), glutathione reductase, γ-glutamyl p-nitroanilide, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, potassium iodide, sodium tartrate, copper sulphate, bromo cresol green, hydrogen peroxide solution, phenazine methosulphate, glycylglycine, magnesium chloride, guaiacol, sulfosalicylic acid, sodium azide, reduced nicotinamide adenine dinucleotide (NADH), sodium hydroxide, and trichloroacetic acid (TCA) were purchased from Sigma Chemicals Co. USA.

Nonylphenol (IUPAC name: 4-(2,4-dimethylheptan-3-yl) phenol; CAS no: 84852-15-3; Purity: 99%) and Aucubin (IUPAC name: 5-Hydroxy-7-(hydroxymethyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-1-yl β-D-glucopyranoside: CAS no: 479-98-1; Purity: ≥ 98.0%) were purchased from Sigma Ald (Germany). All other chemicals used in the study were of analytical grade, such as dimethyl sulfoxide (DMSO), sodium pyrophosphate buffer, Na₃PO₄ buffer, phenazine methosulphate, glacial acetic acid, NADH, NADPH, sodium acetate buffer, N, N-diethyl-para-phenylenediamine, Ellman’s reagent, 5,5-dithiobisnitrobenzoic acid (DTNB), ascorbic acid, trichloroacetic acid and trichlorobarbituric acid were bought from Sigma Aldrich, Germany. Phosphate buffer saline (PBS) and H₂O₂ were purchased from Thermo Fisher, Germany.