Blasticidin solution

The RSV subgenomic replicon system was licensed from Apath (Apath, NY, USA)^{49 (link),50 (link)}. The BHK-derived cell line (APC-126) containing the stable RSV A2 replicon was cultured in DMEM/Hams F-12 medium supplemented with 10% (v/v) FBS (Gibco), 1% (v/v) penicillin (10,000 units/mL) and streptomycin (10,000 µg/mL) solution (Gibco), 1% (v/v) MEM non-essential amino acids solution (Gibco), 5% (v/v) Tryptose Phosphate Broth solution (29.5 g/l) (Sigma-Aldrich) and 10 µg/ml of blasticidin solution (Invivogen). Cells were maintained at 37 °C in a humidified 5% CO₂ atmosphere. 5000 cells were plated in 96-well clear plates (Corning) in 100 µl/well of medium. On the following day, the tested compound was solubilized in 100% DMSO before being transferred in duplicate to the culture plate with 9 points 3-fold serial dilution starting at 10 µM for EC₅₀ determination or 100 µM for CC₅₀ determination (100 µl final volume; 1% DMSO final concentration). Cells were incubated with compounds for 3 days at 37 °C in a 5% CO₂ atmosphere before measurement of the luciferase readout (Promega Renilla Glo assay, 100 µl/well of reagent). Cell viability (CC₅₀ and CC₉₀) was measured with a CellTiter-Glo cell proliferation assay (Promega, 100 µl/well of reagent). Results were expressed as EC₅₀ and EC₉₀ values as well as CC₅₀ and CC₉₀ values.

TMPRSS2-expressing Vero E6 (VeroE6/TMPRSS2) cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB1819) and maintained in low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum (FBS) (Biowest, Bradenton, France) and G418 (Nacalai Tesque, Kyoto, Japan). HEK293-C34 cells were gifted by Y Matsuura at Osaka University and maintained in high-glucose DMEM (Nacalai Tesque) with 10% FBS and 10 μg/ml blasticidin (solution) (InvivoGen, California, USA), and the exogenous expression of ACE2 and TMPRSS2 was induced by the addition of doxycycline hydrochloride (1 μg/ml) (Sigma-Aldrich). All the above cells were cultured at 37°C under 5% CO₂.