Quercetin

Quercetin was acquired from Tokyo Chemical Industry (Tokyo, Japan) and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to prepare the stock solution. All remaining reagents were procured from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies obtained from Santa Cruz (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were utilized: anti-p53 (C-11), anti-cyclin D (M-20), anti-cyclin E (M-20), anti-cdk4 (C-22), anti-cdk6 (DSC-90), anti-Bax (P-19), anti-Bcl-xL (H-5), anti-caspase 3 (L-18), anti-cleaved caspase 3 p11 (h176), anti-caspase 8 p18 (D-8), anti-caspase 9 (96.1.23), anti-cleaved caspase 9 p10 (h331), anti-cytochrome c (A-8), anti-GM3 synthase (T-19), anti-GD3 synthase (B-11), and anti-β-actin (C-4). Additional antibodies (anti-Bid, anti-PARP, anti-MTCO1, and anti-tubulin β) were sourced from Cell Signaling Technology (Danvers, MA, USA) and Novus Biologicals (Novous Biologicals, LLC, Centennial, CO, USA).

The extract of ES was dissolved in MeOH into 0.1 mg/ml. Gallic acid (Sigma aldrich chemie GmbH, Germany), protocatechuic acid (Hwi analytik GmbH, Germany), quercetin (Tokyo Chemical Industry, Tokyo, Japan) and kaempferol (Santa Cruz Biotechnology Inc., USA) were dissolved in MeOH for analysis, either. HPLC was performed on an Agilent 1100 system (Agilent Technologies, Waldbronn, Germany) with a photodiode array detector DAD (G1315D) and Agilent 1100 series quard pump (G1311A), and an Agilent 6410 Triple Quadrupole LC/MS mass spectrometer (Agilent Technologies, Waldbronn, Germany) coupled with an ESI (electrospray ionization) interface and an ion trap mass analyzer. The ESI (electrospray ionization) source was operated in negative ionization modes. Analysis of included compounds were performed under the following conditions: column, TSK-gel ODS-80Ts (Tosoh Co., Tokyo, Japan 4.6 mm X 150 mm); mobile phase, 0.1% formic acid (solvent system A) and CH₃CN (solvent system B) in a gradient mode (B from 20 to 80% in 30 min); sample injection, 5 μl; flow rate, 0.5 ml/min; temperature, 30 °C, UV wavelength, 254 nm and 350 nm. High-purity nitrogen was used as dry gas at a flow rate at 10 L/min, gas temperature at 300 °C; fragmentor voltage 150 V. Nitrogen was used as nebulizer at 30 psi and capillary voltage, ±4000 V.

Epigallocatechin, myricetin, fisetin, quercetin, kaempferol, galangin, naringenin, and Epigallocatechin gallate were purchased from the Tokyo Chemical Industry Co. (Tokyo, Japan). Lipofectamine 3000, CellROX Orange, and CellROX Deep Red were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and the autophagy detection reagent, DALGreen, was purchased from Dojindo Laboratories (Kumamoto, Japan).

Kaempferol, fluvastatin, mevalonate, lipoprotein-deficient serum (LPDS), and U0126 were purchased from Sigma. Quercetin, apigenin, and luteolin were purchased from Tokyo chemical industry. Kaempferol, Quercetin, apigenin, luteolin, and U0126 were dissolved in dimethylsulfoxide (DMSO), and Kaempferol was prepared at the time of use. In this study, the final DMSO concentration in the cultured medium was 0.1%. DiI-labeled LDL was obtained from Molecular Probes.

Standard compounds for LC-MS including rutin, kaempferol, and quercetin were purchased from TCI Chemicals (Tokyo, Japan). p-Nitrophenyl-α-D-glucopyranoside (p-NPG, ≥98%) and acarbose were from TCI Chemicals (Tokyo, Japan). α-Glucosidase enzyme (Saccharomyces cerevisiae, lyophilized powder, 10 U/mg) were purchased from Sigma Aldrich (St. Louis, MO, USA). Dimethylsulfoxide (DMSO) was purchased from RCI Labscan (Bangkok, Thailand).