2.1 Cell cultures and treatments with MDM2 inhibitors

A549-hACE2 cells were a kind gift of Professor Arnaldo Caruso and Professor Francesca Caccuri (University of Brescia, Italy) and were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Carlo Erba, Cornaredo, IT) supplemented with 10% fetal bovine serum (FBS, Gibco, Invitrogen Corporation, NY, United States) and 1% Penicillin-Streptomycin-Glutamine 100X (Sigma-Aldrich, Darmstadt, Germany). Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂ and trypsinized every 2-3 days for passages or experiments. Cultures were routinely checked for mycoplasma contamination.

The day before the experiments, cells were seeded at a density of 8 × 10⁴ cells/mL/well in 12 well plates. The following day, cells were treated with Nutlin-3 (Cayman Chemicals, Michigan, United States) or RG-7112 (Selleckchem, Planegg, Germany) at the predetermined concentrations of 0.1, 1, and 2.5 µM. Cells left with complete medium (untreated, Unt) or treated with dimethyl sulfoxide (DMSO) as drug vehicle were used as internal controls. After 24 h of treatment, cells were harvested for cell cycle and cell growth, apoptosis and western blotting analyses, or treatments were replaced in complete medium supplemented with 5% fetal bovine serum and cells were incubated for 48 h (reaching 72 h of treatment) and then harvested for analyses. Some cultures were infected with SARS-CoV-2 as described in the following paragraphs. The experimental workflow is represented in Supplementary Figure S1.