N3510

Cell viability was measured using WST-1 Cell Proliferation Assay (Abcam, USA ab65473). A total of 1500 cells were seeded onto a 96-well plate and were seated for 24 h before drug treatment with different concentrations of niclosamide (Sigma-Aldrich, USA N3510) and nitro-deficient niclosamide: 5-chloro-N-(2-chlorophenyl)-2-hydroxy-benzamide (Selena Chem, Ukraine SEL10044108) for 48 h. Cells were subsequently recovered in McCoy’s 5A media for 7 d. WST-1 reagents were prepared as recommended by the manufacturer’s protocol. Then, 10 µL of WST-1 reagent was added to each well and cells were incubated with WST-1 reagent at 37 °C and 5% CO₂ for 1 h. Absorbance readings were measured at 440 nm using BioTek 800 TS absorbance reader.

All drugs were prepared to 10 mM stock solutions in 100% DMSO (Sigma) for niclosamide (N3510, Sigma) and ivermectin (I8898, Sigma), or water for chloroquine (HY-17589, MCE) and stored at − 80 °C. The drugs were diluted to the working concentrations in 2%FBS-MEM or 2%FBS-DMEM/F12 for the experiments in Vero E6 or Calu-3 cells, respectively. The final concentration of DMSO was 0.5% in all experiments.