Figure 3.

Synergistic inhibition of AYn1 and AYp28 peptides on pseudovirus invasion into HEK293T/hACE2 cells. (a) Pseudovirus at 10¹⁰/mL were incubated with AYn1 at 0 to 27.04 µM for 2 hours at 37ºC, followed by further respectively incubating with HEK293T/hACE2 cells in 96-well plate for 48-72 hours at 37ºC. (b) HEK293T/hACE2 cells at 30,000 cells/well were plated in 96-well plates and incubated with AYp28 at 0 to 27.04 µM at 37ºC for 2 hours, followed by adding the pseudovirus at 10¹⁰/mL for 48-72 hours at 37ºC. (c) For synergistic assay, the neutralized pseudovirus (preincubated with 0 to 27.04 µM of S-protein neutralizing peptide AYn1 for 2 hours at 37ºC) were added into HEK293T/hACE2 cells pretreated with 5 µM ACE2 protecting peptide AYp28, and further incubated for 48-72 hours at 37°C. Luciferase activity was measured. The results are representative of three independent experiments, with each condition triplicated and presented as the mean ± SD of inhibition of the pseudovirus. (d) Inhibition effect of peptidic cocktail on pseudovirus invasion in HEK293T/hACE2 cells was further confirmed by confocal microscopy. The fluorescence of pseudovirus (green), peptides (red) and nuclei (blue) were observed. Scale bar = 10 µm.