Hygromycin

All the primary cells in this study were cultured in EpiCM (4101, Sciencell (San Diego, California, USA)), but we modified the kit instruction with the replacement of 2% FBS instead of 10% FBS to culture all single cell clones including the epithelial clone L-2F7 and fibroblast clone L-G33. As immortalized gene vectors harbored antibiotic resistance genes, hygromycin (60224ES03, Yeasen) and blasticidin (60218ES10, Yeasen (Shanghai, China)) were selected at concentrations of 200 and 10µg/ml, respectively. The gallbladder cancer cell (GBC) lines NOZ, GBC-SD, ZJU-0430, and OCUG1 were authenticated by Short tandem (STR) repeat and tested if mycoplasma-free. The four GBC cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) (SH30243.01, Hyclone (Logan, Utah, USA)) with 10% FBS and 1% penicillin–streptomycin solution (60162ES76, Yeasen (Shanghai, China)). All the cells were digested by a trypsin solution (40127ES60, Yeasen) before passaging or being frozen with a cell-saving buffer (C30100, NCM (Suzhou, Jiangsu Province, China) Biotech).

Hela‐MMTV‐Luc reporter cells were established by transfecting plasmid pGL4.36[luc2P MMTV Hygro] into Hela cells with lip3000 transfection reagent. For generating a cell line with stable transgene expression, transfected cells were selected by co‐culture with 0.4 mg mL⁻¹ Hygromycin (YEASEN, Shanghai, China). The agonist activities of the tested compounds toward GR were determined in Hela cells which were stably transfected with construct (MMTV‐Luc) by measuring the upregulation of firefly luciferase activity. Briefly, the day prior to assay, cells stably expressing MMTV‐luc were diluted and plated at 1 × 10⁴ cells per well in 96‐well white plates in DMEM medium (5% CSS). Then, cells were treated with the gradient concentrations of the tested compounds for 18 h and then luciferase activity was measured with the One‐Lumi Firefly Luciferase Assay Kit (Beyotime; #RG055M). Bioluminescence was measured with Synergy H1 (BioTek). Control wells with DMSO or DEX at 10 µm were included on each plate to define the 0% and 100% activation effects, respectively. Raw data were transformed to % effect using the following equation:
$$\%\mathrm{effect}=100\times \frac{x-\min }{\max -\min }$$