Quercetin aglycone

Baker yeast α-glucosidase (EC 3.2.1.20), and porcine pancreas α-amylase (EC 3.2.1.1) were from Sigma-Aldrich (St. Louis, MO, United States). Phenolic standards (gallic acid, vanillic acid, caffeic acid, ferulic acid, p-coumaric acid, cyanidin chloride, and quercetin aglycone), carotenoid standards (lutein, zeaxanthin, β-cryptoxanthin), and the Folin–Ciocalteu reagent were from Sigma-Aldrich. The (±)-6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxilic acid (Trolox), and the 2,2-diphenyl-1-picrylhydrazyl (DPPH˙), and 2–2′-azino-bis(3ethylbenothiazoline-6-sulfonic acid) (ABTS^·+) radicals were purchased from Sigma-Aldrich. Pyridine, phenyl-β-d-glucopyranoside, methoxyamine hydrochloride, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and methyl undecanoate were from Sigma-Aldrich.

The UM cell line 92.1 was kindly provided by Prof. Martine J. Jager (Leiden University Medical Center, Leiden, the Netherlands), in whose laboratory this cell line was established [68 (link)]. The 92.1 cells were authenticated by the profiling of short tandem repeats in previous studies [46 (link),69 (link)] and harbor the Q209L-activating mutation in GNAQ [46 (link),69 (link)]. The cells were grown under normoxic conditions at 37 °C with 5% CO₂ in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin (Life Technologies, Darmstadt, Germany) and passaged weekly via trypsinization.
Quercetin aglycone (Sigma-Aldrich, Darmstadt, Germany) was reconstituted in DMSO at 50 mM, stored as aliquots at −20 °C under light protection, and diluted in the culture medium at the indicated concentrations for the subsequent assays. The solvent controls were incubated with the culture medium that was supplemented with DMSO at the same volumes of quercetin. An additional group of cells were incubated in a low-serum medium with 0.5% FBS as a control for growth factor deprivation.

Extraction solvents, including methanol and acetic acid, were purchased from Fisher Scientific (San Jose, CA., USA). Hydrochloric acid, kaempferol (>90% purity), and quercetin aglycone standards were purchased from Sigma Aldrich (St. Louis, MO., USA). All solvents for ultra-high-performance liquid chromatography-photodiode array (UHPLC-PDA) and mass spectrometry (LC-MS/MS) were purchased from Fisher Scientific (USA).