Ngi 1

For a list of antibodies and guide RNAs (gRNAs) used in this study please see Table S2. NGI-1 was purchased from Sigma-Aldrich and C19 (N-(5-(Morpholinosulfonyl)-2-(pyrrolidin-1-yl)phenyl)benzamide) was purchased from Mcule.

Wild-type and gene modified clones of the MCF7 human breast carcinoma cell line were maintained in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine, 10000 units/ml penicillin, 10 mg/ml streptomycin (all from Biological Industries, Beit HaEmek, Israel), and cultured at 37 °C in a humidified incubator supplied with 5% CO2. Glucose free medium was supplemented with 4 mM L-Glutamine, 10% Fetal Bovine Serum, 10000 units/ml penicillin and 10 mg/ml streptomycin. For short term experiments a dialyzed Fetal Bovine Serum (Biological Industries) was used. ROCK inhibitor Y27632 (ROCKi) was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and used at a final concentration of 10 µM. PEPCK-M inhibitor (iPEPCK-2) was produced in the laboratory of Dr. Carmen Escolano and used at a final concentration of 5 µM. Inhibitors of glycosylation, NGI-1 and Tunicamycin used at final concentration 10 µM and 3 µg/ml, respectively, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Thapsigargin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used at a final concentration 0.1 µM. Taxol (Teva, Jerusalem, Israel) was used at final concentration 1 µM.

Cells were treated with either DMSO or several N-glycosylation inhibitors (from DMSO stock solutions; DMSO final concentration never exceeding 2%) in DMEM–2% FBS. Inhibitors used were tunicamycin (Sigma), NGI-1 (Sigma), DNJ (Cambridge Bioscience), miglitol (Stratech Scientific), miglustat (Cambridge Bioscience), acarbose (Fisher Scientific), NN-DNJ (Cambridge Bioscience), celgosivir (Sigma), DMJ (Tebu-Bio), and swainsonine (Cambridge Bioscience). Cells were then infected by incubation with SARS-CoV-2 P4 supernatant with the corresponding inhibitor for 30 min at 37°C and 5% CO₂ at MOI of 0.001 (Vero E6 for plaque assay), MOI of 0.05, or MOI of 0.1 (Vero E6 and HEK293^ACE-2 for immunofluorescence assay, respectively). Cells were washed and left in DMEM–2% FBS and N-glycosylation inhibitor at 37°C and 5% CO₂ for 48 h (Vero E6 plaque assay) or 24 h (immunofluorescence). For toxicity assays, cells in 96-well plates were treated with variable concentrations of inhibitors in DMEM–2% FBS in triplicate, and after 48 h, an alamarBlue 10× solution (Invitrogen) was added and incubated for 4 h at 37°C before measuring well fluorescence intensity at 590 nm using a FLUOstar Omega plate reader (BMG Labtech).