SARS-CoV-2 Infection Assay in Calu-3 and Vero E6 Cells

Calu-3 cells^{52 (link)} (ATCC HTB-55) were cultured according to ATCC recommendations. All experiments were performed in these cells below passage 15. Vero E6 cells (ATCC CRL-1586; used for SARS-CoV-2 plaque assay) were grown in minimum essential medium (MEM) supplemented with 10% FBS, 1 mM sodium pyruvate and 0.1 nM non-essential amino acids, and used at passage <40. All cells were expanded in a T175 flask with 5% carbon dioxide at 37 °C. Cell density was kept between 0.25 and 2 million cells per ml. Vero E6 cells (used for TMPRSS2 proteolytic activity screening and IC₅₀ determination) were maintained in Eagle’s minimum essential medium (EMEM) containing 10% foetal bovine serum, 2 mmol l⁻¹l-glutamine, 100 IU ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Cm was obtained from MilliporeSigma. The SARS-CoV-2 nucleocapsid antibody (HL344) (GTX635679) was provided by Genetex; mouse anti-dsRNA antibody (J2-1904) was purchased from Scions English and Scientific Consulting^{33 (link)}; Hoechst 33342 and secondary antibodies goat anti-mouse IgG Alexa Fluor 488 (A11001) and goat anti-rabbit IgG Alexa Fluor 555 (A21428) were obtained from Invitrogen. Cell lines were screened for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza).