Glycosylation Profiling and SARS-CoV-2 Spike Protein Binding

Cells treated with compounds or DMSO control were stained with biotinylated lectins (Vector Laboratories) (VVA, PNA 0.2 μg/mL; Pan-Lectenz, 2,3-Lectenz, GSL II, PHA-L, GNL, DSL, RCAI, ECL, LCA, PHA-E, WGA 1 μg/mL) or mouse mAbs to Tn (1E3)^{44 (link),99 (link)}, Tn-MUC1 (5E5)^{65 (link)}, FXYD5 (6C5 and NCC-MC53)^{64 (link)} diluted 1:5000 in PBA (PBS with 1% (w/v) BSA) for 1 h at 4 °C. For SARS-CoV-2 spike protein binding, NSC80997, DMSO control or Heparinase mix (2.5 mU/mL HSase II, and 5 mU/mL HSase III; IBEX) treated cells were incubated with recombinant SARS-CoV-2 biotinylated spike protein S1/S2 (20 µg/mL) for 30 min at 4 °C. Spike protein was produced and biotinylated as previously described^{71 (link)}. Cells were washed with PBA and incubated with streptavidin conjugated to Alexa Fluor 488 or 647 (Invitrogen), FITC-conjugated rabbit anti-mouse immunoglobulins (Dako), or Goat anti-mouse IgG, Alexa Flour 488 (Invitrogen) diluted 1:2000 in PBA, respectively. Cells were washed twice and resuspended in PBA and analyzed using a SA3800 spectral analyzer running the SA3800 software (SONY) or a FACSCalibur instrument running BDFACStation software (BD Bioscience). Cells were gated to exclude dead cells and doublets (Supplementary Fig. 4B) and data was analyzed using FlowJo software (FlowJo, LCC).