Dulbecco modified eagle medium (dmem)

Huh7.5.1 [28 (link)], GP2-293 (Clontech Laboratories, Inc., Mountain View, CA, USA), HEK293T (ATCC^® CRL-3216^™, Manassas, VA, USA), and HepG2/C3A (ATCC^® CRL-3581) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS, Minneapolis, MN, USA). In vitro transcription was performed from p6/luc, a HEV replicon that contains cDNA of the genomic RNA of Kernow-C1, a genotype 3 strain, with an insert encoding Gaussia luciferase, replacing the 5′ portion of ORF2 [29 (link)]. The luciferase is secreted out of the cells after synthesis. The Kernow-C1 strain p6 was used to infect the HepG2/C3A cells at a multiplicity of infection (MOI) of 1. The infected HepG2/C3A cells were passaged five times to produce stably infected cells.

Human triple-negative (MDA-MB231 (#92020424) and HS578T (#86082104)) and oestrogen receptor-positive breast cancer cell lines T47D (#85102201) and ZR-75-1 (#87012601) were purchased from European Collection of Cell Cultures (ECACC) General Cell Collection. Cell lines were cultivated at 37 °C in a humidified atmosphere containing 5% CO2 and used until passage number 25.
HS578T cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; #10–013-CV, Corning, Corning, NY, USA) supplemented by 10% fetal bovine serum (FBS; #P40-37500 PAN-Biotech, Germany) and 1% penicillin/streptomycin (#10378016, Thermo Fisher Scientific, Waltham, MA, USA). MDA-MB231, T47D, and ZR-75-1 breast mammary gland carcinoma cell lines were maintained in RPMI-1640 base medium (#BE12-702 F, Lonza Biosciences; Basel, Switzerland) using fetal bovine serum (FBS; #P40-37500 PAN-Biotech, Germany) in a final concentration of 10% and 1% penicillin/streptomycin (#10378016, Thermo Fisher Scientific, Waltham, MA, USA).
Three times a week, the cell culture medium was replaced with a fresh complete medium. When cells reached 90% confluence, they were detached from the bottom of the flask using 0.05% Trypsin-EDTA (#25300062, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Microscopic control and imaging were done by EVOS M7000 imaging system using a ×10 objective.
In experimental settings, cells were kept in steroid-free media for 48 h before plating. Steroid-free media was prepared by using charcoal-stripped FBS as previously reported^{13 (link)}. Then, cells were plated on the six-well tissue culture plates (maintaining complete or steroid-free conditions). Seed numbers on the six-well plate were 50,000 and 100,000 cells/well for HS578T, T47D and MDA-MB231, ZR-5-1, respectively. On 24-well plates we seeded 20,000 and 400,000 cells/well regarding HS578T, T47D and MDA-MB231, ZR-5-1, respectively. Cells were treated with 100 nM dexamethasone and mifepristone^{13 (link)}. All experiments were repeated at least three times, with one technical replicate for each sample.
Three-dimensional spheroid formation was induced by ultra-low attachment of six-well plates (07–200-601, Corning, Corning, NY, USA). Experiments were done following a 3–4-day process of spheroid induction.

Human hepatoma cells (HepG2) were purchased from American Type Culture Collection (HB-8065, ATCC, Manassas, VA, USA) and were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Corning, Corning, NY, USA). DMEM was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). Cells were grown at 37 °C with 5% CO₂. To passage cells, the cells were first rinsed with sterile phosphate-buffered saline (PBS) (Gibco). Then, 0.25% Trypsin-EDTA (Gibco) was added to the cells and incubated at 37 °C for approximately 10 min or until the cells lifted. Fresh cell culture media were then supplemented back in, and the cells were replated.