Fig. 6

EGCG blocks SARS-CoV-2 S binding to ACE2. a–d A549 cells were transfected with pCAG-hACE2 plasmid. Twenty-four hours later, the cells were incubated with medium containing His-tagged SARS-CoV-2 S (full-length S, S1, S2 and RBD, 1 μg/mL) with or without EGCG (50 μM) for 6 h, and subsequently incubated with goat anti-ACE2 and mouse anti-His-Tag antibodies, following stained with donkey anti-goat IgG antibody conjugated with Alexa Fluor 594 and donkey anti-mouse IgG antibody conjugated with Alexa Fluor 488. Nuclei were stained with Hoechst. All images were obtained by confocal microscopy (Nikon A1R, Nikon, Japan). The scale bar in each panel indicates 25 μm. e VeroE6 cells were incubated with medium containing His-tagged SARS-CoV-2 S (full-length S, S1, S2 and RBD, 1 μg/mL) with or without EGCG (50 μM) for 6 h, and then washed 3 times with PBS to remove unbound protein. Cell lysate was subsequently subjected to western blot analysis to detect the binding of His-tagged S to ACE2. GAPDH was set as internal control of western blot. (f–h) SARS-CoV-2 S (full-length S, S1 and RBD, 1 μg/mL) was pre-mixed with EGCG at the indicated dose, and the pre-mixture was then incubated with VeroE6 cells for 6 h, free S protein was removed with PBS triple washing. Western blot analysis was performed to assess the binding of S protein to ACE2. (i) Immobilized hACE2 protein (Fc tag) was pre-coated at 2 μg/mL at 4 °C overnight, and threefold serially-diluted His-tagged SARS-CoV-2 full-length S (500–0.686 ng/mL) and RBD (100–0.137 ng/mL) were used to test the binding affinity to ACE2