Methanol

Phenylephrine hydrochloride (Sigma, cod. P6126), Acetylcholine chloride (Sigma, cod. A6625), Nω-Nitro-L-arginine methyl ester hydrochloride (Sigma, cod. N5751), charybdotoxin (Sigma, cod. C7802), indomethacin (Sigma, cod. I7378), apamin (Sigma, cod. A1289), Dihydroethidium (Sigma, cod. D7008), 2,2-Diphenyl-1-picrylhydrazyl (Sigma, cod. D9132), Folin-Ciocalteu’s (Sigma, cod. 47,641), ascorbic acid, gallic acid, from Sigma-Aldrich®, 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) from Invitrogen Molecular Probes™ (cod. D1306) and Dako Fluorescence Mounting Medium (cod. S3023) Na₂CO₃, NaCl, KCl, KH₂PO₄, NaHCO₃, C₆H₁₂O₆, CaCl₂, MgSO₄, ethylenediaminetetraacetic Acid (EDTA), ethanol, methanol, from Vetec®.

Yeast extract, peptone, biotin, dextrose, agar, peroxidase-conjugated anti-rabbit secondary antibody, Pefabloc SC (4-(2-aminoethyl)-benzenesulfonyl fluoride), sorbitol, EDTA (ethylenediaminetetraacetic acid) and DAB (3,3’-diaminobenzidine) substrate kit were purchased from Sigma (Missouri, USA). Glycerol and methanol were supplied by VETEC (Rio de Janeiro, Brazil). Pichia pastoris X-33, pPICZαA, and Zeocin were purchased from Invitrogen (California, USA). Restriction enzymes and PNGase F were furnished by New England Biolabs (Massachusetts, USA). Electroporation cuvettes, TMB (3,3’,5,5’-tetramethylbenzidine) EIA substrate kit and low-range SDS-PAGE standards were obtained from Bio-Rad Laboratories (California, USA). Trypsin was purchased from Promega (California, USA). Pierce Glycoprotein Staining kit, DMEM (Dubelcco’s Modified Eagle Medium), and fetal bovine serum were purchased from Thermo Fisher Scientific (Massachusetts, USA).

The solvents acetonitrile and formic acid were obtained from Tedia (HPLC grade, Radnor, PA, USA), and ultrapure water was obtained from the Milli-Q^® Plus apparatus by Millipore (Billerica, MA, USA). Methanol, ethanol, isopropanol, polysorbate 80 (Tween^® 80), dimethyl sulfoxide (DMSO), and sodium hydroxide were obtained from Vetec (Duque de Caxias, Brazil), and medium chain triglycerides (MCT) and egg lecithin (Lipoid^® E80) were purchased from Lipoid GmbH (Ludwigshafen am Rhein, Germany). Monobasic potassium phosphate was obtained from Dinâmica (São Paulo, Brazil), and NBD-PE [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt] fluorescent-labelled phospholipid was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Roswell Park Memorial Institute 1640 broth medium (RPMI-1640), 3(N-morpholino) propane sulfonic acid (MOPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and itraconazole were obtained from Sigma-Aldrich (St. Louis, MA, USA), and potato dextrose agar (PDA) was purchased from Acumedia (San Bernardino, CA, USA). Tissue-Tek^® O.C.T.™ was purchased from Sakura Finetechnical Co. (Tokyo, Japan). Log P (calculated log P) values of the coumarins were assigned using SwissADME http://www.swissadme.ch/ (accessed on 10 March 2023).

The biomass quantification test was performed according to Marcos-Zambrano et al. (2014) (link), with some modifications. Briefly, biofilms were formed in 96-well plates (Kasvi) at different incubation times. The resulting supernatants were removed and the remaining biofilms were washed with PBS. Two hundred microliters of methanol (Vetec) were added to each well for 15 min. The methanol was discarded and the plates were dried at room temperature for 45 min. After drying, 200 μL of 0.1% violet crystal solution (Dinamica) was dispensed into the wells for 20 min. The resulting supernatant was carefully aspirated and the wells were washed with distilled water until the complete removal of excess dye. Finally, 200 μL of 33% acetic acid (Synth) was added and plates were read using a spectrophotometer (Biotek Epoch^TM 2 Microplate Spectrophotometer) at a wavelength of 570 nm. The resulting absorbance values were directly proportional to the amount of biomass.