Foetal bovine serum (fbs)

Hep G2 cells, HuH-7cells, human liver vascular endothelial (TMNK-1) cells and human bile duct epithelial (MMNK-1) cells were obtained from the Japanese Center Research Bank and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Wako) supplemented with 10% foetal bovine serum (Corning) and 100 units/ml of penicillin-streptomycin (Wako). Cells were grown in an incubator at 37 °C and supplied with 5% CO₂. Mouse bone marrow cells were isolated from the femurs and tibias of 8-week-old C57BL/6NcrSlc male mice (Japan SLC) using previously described method^{24 (link)}. Isolated cells were cultured in DMEM (containing 10% fetal bovine serum and 100 units/ml of penicillin-streptomycin) at 37 °C in a 5% CO₂ incubator. All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committees of Laboratory Animal Experimentation (Animal Research Center of Yokohama City University, Yokohama, Japan).

All cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). MCF7, T47D, ZR-751, BT474, SKBR3, MDA-MB-453, and MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Corning, NY), and HCC1937, HCC38, and BT20 cells were cultured in RPMI medium (Corning). MCF10A cells were maintained in DMEM/F12 (Invitrogen, CA, USA) supplemented with 20 ng/mL epidermal growth factor (Peprotech, London, UK), 0.5 mg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 100 ng/mL cholera toxin (Sigma-Aldrich), and 10 μg insulin (Sigma-Aldrich). All media were supplemented with 10% foetal bovine serum (Corning) and 1% penicillin/streptomycin, and maintained in a humidified 5% CO₂ incubator at 37 °C. Radioresistant CD44^high/CD24^low MCF7 cells were established using a previously described method^{24 (link),25 (link)}. In brief, CD44⁺/CD24⁻ subpopulations from MCF7 cells were isolated according to their surface markers with flow cytometry using a FACS Aria II system (BD Biosciences, San Diego, CA, USA). A radioresistant phenotype was determined using colony and sphere forming assays in response to irradiation. Cells were irradiated using a ¹³⁷cesium (Cs) ray source (Atomic Energy of Canada, Mississauga, Canada) at a dose rate of 3.81 Gy/min. Everolimus (10 nM; Selleckchem, Houston, TX, USA) was used to inhibit the mTOR-S6K1 pathway.

SARS-CoV-2 isolates belonging to B (considered as reference strain in this study), AY.4 (Delta) and BA.1 (Omicron) lineages were incubated with two-fold serial dilutions of serum samples starting at 1:8 dilution in E MEM culture medium (Sigma Aldrich, Merck Life Science, Milan, Italy) supplemented with 1X penicillin/streptomycin (Corning, Glendale, AZ, USA) and 2% foetal bovine serum (Corning) in 96-well plates. Virus (100 TCID50) and serum mixture was incubated at 37°C for 1 hour. After this incubation 22,000 cells per well were added and incubated at 37°C for 5 days. The neutralization titer was calculated and expressed as microneutralization titer 50 (MNT50), i.e., the serum dilution capable of reducing the cytopathic effect to 50%.