Penicillin streptomycin solution

Vero cells (ATCC CCL-81, Manassas, Virginia, United States) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L glucose (Microtech, Naples, Italy) supplemented with a penicillin/streptomycin solution (Himedia, Naples, Italy) and 10% Fetal Bovine Serum (FBS, Microtech). HSV-1 (strain SC16), containing a lacZ gene driven by the cytomegalovirus IE-1 promoter to express beta-galactosidase, was grown on a Vero cell monolayer, as previously reported [29 (link)]. An acyclovir-resistant HSV-1 strain was selected in Vero cells receiving suboptimal acyclovir (ACV, Sigma-Aldrich) treatment.

African green monkey kidney epithelial cell lines (Vero CCL-81, Manassas, VA, USA) were acquired from the American Type Culture Collection (ATCC), cultivated in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose (Microtech, Naples, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Microgem, Naples, Italy), 2 mM L-glutamine (Microtech), and 100 IU/mL of penicillin–streptomycin solution (Himedia, Naples, Italy), and maintained at 37 °C in a humidified atmosphere with 5% CO₂. All described viral strains were propagated on Vero cell lines: HSV-1 (strain SC16), HSV-1expressing the VP22-green fluorescent protein, HSV-2 (strain 333), poliovirus type 1 (PV-1 strain CHAT), human coronavirus 229E (HCoV-229E strain VR-740), and SARS-CoV-2 (strain VR-PV10734; kindly provided by Lazzaro Spallanzani Hospital, Rome, Italy) [58 (link)]. The experiments involving SARS-CoV-2 were carried out in a biosafety level 3 (BSL-3) containment laboratory (University of Salerno) according to World Health Organization (WHO) recommendations.

Verapamil hydrochloride, piperine, vincristine sulfate, rhodamine 123 (Rho123), dimethyl sulfoxide (DMSO), RIPA buffer, and protease inhibitor cocktail were purchased from Sigma-Aldrich, USA. Colchicine, Dulbecco’s Modified Eagle Medium (DMEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypsin-ethylene diamine tetra acetic acid (Trypsin–EDTA) solution, penicillin–streptomycin solution, phosphate-buffered saline (PBS), acridine orange (AO), ethidium bromide (EB), 4′,6-Diamidino-2-Phenylindole, dihydrochloride (DAPI), Bradford reagent and fetal bovine serum (FBS) were obtained from HiMedia (Mumbai, India). Nitrocellulose membrane was purchased from GE healthcare, USA. Anti-P-gp (D-11), and anti-tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).