Dulbecco modified eagle medium (dmem)

Vero E6 cell lines (African green monkey kidney epithelial cells, ATCC^® CRL‐1586™) were cultured in Dulbecco's modified Eagle's medium (DMEM; Bio‐Concept, Salem New Hampshire, USA) supplemented with 10% fetal bovine serum (FBS; Gibco™, Thermo Fisher, Waltham, MA, US). Vero E6 cells were cultured to an optimum monolayer confluence and infected with SARS‐CoV‐2 2019‐nCoV/Italy‐INMI1, the first viral strain isolated in Italy in January 2020 of which the complete sequence was submitted to GenBank (ID: MT066156) and is available on GISAID website (BetaCoV/Italy/INMI1‐isl/2020: EPI_ISL_410545). SARS‐CoV‐2 2019‐nCoV/Italy‐INMI1 was cultured in a DMEM medium supplemented with 5% FBS to a low specific Multiplicity Of Infection (MOI) of 0.05 and incubated at 37°C for 46–50 h in roller bottles (surface of 850 cm²). A single batch virus which was cultured without fetal serum to better analyze the electrophoretic pattern of the proteins was also produced.
SARS‐CoV‐2 isolation and culturing were performed by trained personnel in a Biosafety Level 3 Laboratory (BSL‐3).

HEK Freestyle 293F (HEK293F; Thermo Fisher Scientific) cells were cultured in 15-cm cell culture dishes containing Dulbecco’s modified Eagle’s medium (DMEM; BioConcept) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PenStrep; PAN Biotech) with 5% CO₂ and cultured at 37°C. At 80% confluence, cell medium was exchanged to DMEM supplemented with 2% FBS and 1% PenStrep. The transfection mixture was prepared with Cx32-YFP-twinStrep plasmid and branched polyethyleneimine (Sigma-Aldrich) at a ratio of 1:2 (w/w) with serum-free DMEM. The transfection mixture was added to cells after 5 min of incubation at room temperature. After 48 hours of expression at 37°C and 5% CO₂, the cells were harvested using a cell scraper in buffer A containing 25 mM tris-HCl (pH 8.0) and 150 mM NaCl and collected by centrifugation at 800g and 4°C. The cells were washed with buffer A and collected by centrifugation. The supernatant was discarded, and the cells were frozen at −80°C for further experiments.