HEK 293/hACE2 cells were pretreated with either lysosomotropic agents (endosome acidification inhibitor NH4Cl and bafilomycin A; PIKfyve inhibitor apilimod and YM201636; calcium channel blocker tetrandrine) or cathepsin inhibitors (cathepsin L inhibitor SID26881509, cathepsin B inhibitor CA-074, and cathepsin inhibitor E64D) for 1 h at 37 °C, then spin-inoculated with pseudovirions in the presence of inhibitor at 1200 × g for 30 min. After overnight incubation, cells were fed with fresh medium without inhibitor. Cells were lysed at 48 h post inoculation and their luciferase activity was measured.