Quercetin

Mouse monoclonal anti-VSV-G (used at 1:1000) and HSV-ICP8 (1:1000) were obtained from Santa Cruz lnc. (Santa Cruz, CA, USA). The antibodies to actin (1:1000), goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP), and goat anti-rabbit secondary antibodies conjugated to HRP (1:10,000) were obtained from Beyotime Biotechnology (Haimen, China). The rabbit polyclonal antibody to phosphorylated (p)-eIF4E (Ser 209, 1:1000) was purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibody to eIF4E (1:1000) was purchased from Proteintech (Wuhan, China). Mouse polyclonal antibody to PEDV-N (1:1000) was purchased from Alpha Diagnostic International (San Antonio, TX, USA). Rabbit polyclonal antibody to AIV-NP (1:2000) was purchased from Abcam (Cambridge, MA, USA). The antibodies to NDV-NP (1:2000) and PRV-gC (1:1000) were preserved in our laboratory. Antibodies were diluted in phosphate-buffered saline (PBS) containing 5% skim milk (pH 7.3). Ribavirin, acyclovir, quercetin, and the mitogen-activated protein kinase (MAPK)-interacting kinase 1 and 2 (MNK1/2) kinase inhibitors CGP57380 were obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Two HBV-replicating cell lines HepG2.2.15 and HepG2.A64 were employed in the study. HepG2.A64 as an ETV-resistant HBV-replicating cell line has been employed previously (Liu et al., 2016 (link); Liu et al., 2018 (link)). Compared to HepG2.2.15 cells, HepG2.A64 cells generated comparable HBV DNA, higher HBsAg but lower HBeAg. The cytotoxicity of LWWL (Shibo Jindu, Zibo, China) and four compounds quercetin, luteolin, wogonin, and kaempferol (purchased from MedChemExpress Co., Ltd., Monmouth Junction, United States) on cells were analyzed using Cell Counting Kit-8 (Dojindo Laborarories, kyushu, Japan) according to the manufacturer’s instructions. The median cytotoxic concentration (CC₅₀) was calculated. The molecular and cellular studies were carried out in Biosafety level-2 (BSL-2) laboratory at Center Laboratory, The Fifth Medical Center of Chinese PLA General Hospital. All manipulations were strictly conducted according to the instructions of the laboratories.

ACS grade solvents (methanol, ethyl acetate, n-hexane, acetone, and chloroform), HPLC grade solvents (ethyl acetate, acetone, and n-hexane) and deuterated solvents (CDCl₃, acetone-d₆, or CD₃OD) for NMR measurements were procured from Merck, Taipei, Taiwan. LPS (endotoxin from Escherichia coli, serotype 0127:B8), Carr (type IV), and quercetin were purchased from MedChemExpress (Monmouth Junction, NJ, USA). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was acquired from Sigma Chemical Co. (St. Louis, MO, USA).

CDDP was purchased from Sigma Aldrich (St. Louis, MO, USA). Primary antibodies against Bcl-2 and GAPDH and all secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). High purity (98.02%) quercetin was obtained from MedChemExpress (Monmouth Junction, NJ, USA).

The PEDV YN144 (GenBank accession No. KT021232) and DR13 (GenBank accession No. JQ023161) strains were used throughout the present study. CCL-81 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 8% fetal bovine serum (FBS) and incubated at 37 °C with 5% CO₂. Virus titers were determined by calculating the 50% tissue culture infectious dose (TCID₅₀) using the Reed–Muench method. Quercetin was purchased from Medchemexpress (Shanghai, China) with a purity of more than 99%. The rabbit polyclonal anti-Hsp70 antibody, mouse anti-β-actin mono-antibody (Mab) and HRP labeled antibodies were purchased from ABclonal (Wuhan, China). The Alexa 488-labeled anti-mouse antibody was purchased from Antgene (Wuhan, China). MAb against the PEDV N protein was purchased from Youlong Biotech (Shanghai, China). MAb against the PEDV S protein was developed by our laboratory.

The human TNBC cell lines MDA-MB-231 and HCC1187, the human mammary epithelial cell line MCF-10A, the mouse TNBC cell line 4T1, the human luminal breast cancer cell lines MCF-7 and T47D, and the human HER2-positive breast cancer cell lines HCC1954 and SKBR3 were procured from ATCC (Manassas, VA, USA) and were sustained in culture medium (MDA-MB-231 in DMEM (Gibco, Grand Island, NY, USA, C11995500BT), other cell lines in RPMI-1640 (Gibco, C11875500BT)) supplemented with 10% fetal bovine serum (FBS, Biological Industries, Cromwell, CT, USA, 04-001-1ACS) and 100 units per ml penicillin/streptomycin (HyClone, Logan, UT, USA, SC30010) at 5% CO₂ and a moderate temperature of 37°C in an incubator. We prepared the culture medium for MCF-10A cells as described previously (12 (link)). The cell lines were free of mycoplasma contamination and were verified by short tandem repeat (STR) profiling. Quercetin and β-sitosterol were purchased from MedChemExpress (MCE, Princeton, NJ, USA, HY-18085 and HY-N0171A). To procure the aqueous extracts of SCH, the shredded herb was boiled with a 10× volume of water for 2 h (in duplicate), followed by freeze-drying of the concentrate. The resultant dry powder was preserved at -20°C. The doses used in the current investigation were aliquoted as an equivalent weight of raw herb per ml.