Serum free media

In order to clone the ctr1, ctr2 and atox1 promoters and characterize their function, yellow catfish were purchased from a local farm (Wuhan, China). HEK293T cells were obtained from the Cell Resource Center in the Fishery College of HZAU. The Lipofectamine 293 and LightShift Chemiluminescent EMSA Kit were purchased from Beyotime Biotechnology (Shanghai, China); Passive Lysis Buffer and Dual-Luciferase from Promega (Minneapolis, MN, USA); and 0.25% trypsin-EDTA, Dulbecco’s Modified Eagle’s Medium (DMEM), Serum-Free Media, Serum-Free Cell Freezing Medium and fetal bovine serum (FBS) from Gibco (Waltham, MA, USA). The Pierce BCA protein assay kit, Trizol reagent and the Reverse Transcription Kit were from Thermo Fisher Scientific (Waltham, MA, USA).

Hybridoma cells were cultured in serum-free media (Gibco) with agitation on a shaker at 100 RPM in a chamber supplemented with 5% CO₂ for 72 h. The conditioned media were collected, and cell debris were removed by centrifugation at 13,000 × g for 30 min. The remaining debris were finally removed by filtration through a 0.22-μm syringe filter (Millipore). The filtrates were purified on a HiTrap™ Protein G HP column (GE Healthcare Life Sciences) using an FPLC system (AKTA purifier, GE Healthcare Life Sciences). The column was equilibrated with PBS buffer at a flow rate of 1 min/ml, and bound proteins were eluted with 50 mM glycine-HCl (pH 2.5) at a flow rate of 3 ml/min. The eluted fractions were neutralized with 1 M Tris-HCl (pH 8.0) and then mixed with a protein-stabilizing cocktail solution (Thermo Fisher Scientific) prior to storage at −20 °C. Isotyping of the purified antibodies was performed using a mouse immunoglobulin isotyping kit (Antagen Pharmaceuticals). The protein concentration was adjusted to 1.0 μg/ml with PBS, and 50 μl of protein solutions were dropped into the sample-loading slots. The isotype was determined by the position of the red line formed.

cDNA for S6K1 and 4EBP1-GFPSpark constructs were obtained from Sino Biological (China); mCherry-raptor, YFP-mTOR, YFP-PRAS40 and FLAG-mTOR from Addgene (USA). EGFP-mTOR, mDsRed-Rheb, EGFP-Rheb and mDsRed-raptor constructs were cloned previously^{21 (link)}. Miniprep and Maxiprep kits purchased from Qiagen (Germany). Rapamycin, L-leucine, L-serine and Ponceau S stain were purchased from Sigma-Aldrich (USA).
HEK293 cell line was purchased from ATCC^® (USA), tested for mycoplasma contamination, and HEK293F cells were provided by Evotec (UK) Ltd. Cell culture dishes (35 mm × 20 mm) were bought from MatTek (USA).
HEK293 and HEK293T cells were cultured in Minimum Essential Media (MEM) supplemented with 10% Foetal Bovine Serum (FBS), 2 mM L-glutamine and 1% Penicillin-Streptomycin (P/S) for HEK293. HEK293F cells were grown in serum-free media (Gibco/Life Technologies, UK). All culture reagents were from Life Technologies. Cells were incubated at 37 °C with 5% CO₂ humidified air in T75 culture flasks (Thermo Fisher Scientific, UK) or in 50 mL tubes (Corning^®). SF9 cells were cultured in SF 900 III media supplemented with 1% P/S at 26 °C.

Vero cells were seeded at 1.2 × 10⁴ cells in 384-well, μClear plates (Greiner Bio-One, Kremsmünster, Austria) 24 h prior to the experiment. Compounds were placed in intermediate 384-well plates containing serum-free media (Gibco, Waltham, MA, USA). The diluted compounds (10 concentrations, 0.05–25 μM) were added to the cell plates in 10 μL volumes (at a final DMSO concentration of 0.5% (v/v)). For viral infection, plates were moved into the BSL-3 containment facility, and SARS-CoV-2 was added to the Vero cells at a multiplicity of infection (MOI) of 0.0125. At 24 h post-infection, the cells were fixed with 4% paraformaldehyde and stained with SARS-CoV-2 nucleocapsid protein, and immunofluorescence analysis was performed. To quantify the number of cells and infection ratios, Columbus software (PerkinElmer, Waltham, MA, USA) was used, and antiviral activity was normalized to the positive (mock-infected) and negative (0.5% DMSO) controls in each assay plate. Concentration–response curves were fitted by sigmoidal models using XLfit 4 in Microsoft Excel or GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, USA). The IC₅₀ values were calculated from curves fitted to the normalized activity dataset. The quality of each assay was controlled by the Z′-factor [25 (link)].