Antibody-mediated cell-cell fusion requires the furin cleavage of spike.

(A) Immunoblots showing Proteinase K-resistant S2’ cleavage product, obtained from WT and R685A spike HEK293T cell lysates, stimulated with 12.5 nM CB6 for 16 hours. Lysates were then treated without or with 10 μg/mL proteinase K for 30 min at 37°C. Blots are representative of two individual repeats; (B) Representative confocal images of 12.5 nM CB6 antibody-stimulated HEK293T cells expressing WT or R685A spike mutant for 16 hours. Anti-LAMP1 and Anti-human IgG (H+L chains) were stained with Alexa fluor 488 and 555 respectively, co-localizations are indicated with white arrows, scale bars are representative of 10 μm. Images are representative of two individual experiments; (C) Immunoblots showing full-length S, cleaved S1, furin and tubulin, collected from HEK293T cells co-expressing 50 nM non-targeting (siControl) or human furin-targeting (siFURIN) siRNAs with full-length spike for 24 hours. Blots are representative of three individual repeats; (D) Representative fluorescent images of HEK293T cells co-expressing WT or Delta spike VOCs, co-cultured with pcDNA4 control or FURIN over-expressing cells stimulated without or with 12.5 nM CB6 antibody for 16 hours, scale bars are representative of 50 μm. Images are representative of two individual experiments; (E) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, hIgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of HEK293T cells expressing spike WT or Delta VOC stimulated with 12.5 nM CB6 antibody for 16 hours. Data are representative of five repeats. (TIF)