Dx600

In the inhibitor studies, some cells were preincubated with DX600 (10 µM; Bachem, Bubendorf, Switzerland) or heparin (200 ug/mL; Sigma, St. Louis, MO, USA) 30 min before spike protein or PSV treatment. The cells were then treated with either the spike proteins or PSVs and processed for the flow cytometric analyses described above.

To reveal the involvement of various endocytosis pathways, A549 cells were preincubated with the following inhibitors 30 min before SARS-CoV-2 admission: amiloride hydrochloride (at a concentration of 100 µM; Sigma, St. Louis, MO, USA); DX600 (10 µM; Bachem, Bubendorf, Switzerland); Gö 6983 (10 µM; Sigma, St. Louis, MO, USA); heparin (200 ug/mL; Sigma, St. Louis, MO, USA); a heparin-binding peptide (WQPPRARI, 100 µM; Genscript, Leiden, The Netherlands); and SPRRAR derived from spikeS1 (100 µM; Genscript, Leiden, The Netherlands). After incubation with these inhibitors, the cells were then treated with SARS-CoV-2 and processed for the flow cytometric analyses as described above.

The ACE2 activity assay was based on the fluorescence of the product generated upon hydrolysis by the enzyme of the substrate Abz-Ser-Pro-3-nitro-Tyr-OH (SPNPT; Bachem E2920). Reactions were initiated by adding 70 μL of the appropriate incubation mixture (substrate, 50 mM Tris-HCl buffer, pH 7.5, 2 μg BSA) to 30 μL of sample. After incubation, the fluorescence intensity was measured with an excitation filter at 355 nm and an emission filter at 410 nm. The inhibition assay for ACE2 activity was performed using DX600 (1 μM, Bachem).