Transfection Efficiency and Pseudovirus Entry Assay

The cultured cells were fixed with 4% paraformaldehyde for 30 min. Samples were incubated with 5% BSA for 1 h. For the transfection efficiency of ACE2, an immunofluorescence assay was performed, as previously described [26 (link)], using an anti-ATPase antibody (EP1845Y, Abcam, Cambridge, UK), a plasma membrane loading control (Abcam), and a primary ACE2 antibody (E-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA) all at a dilution of 1:400, at 4 °C overnight, followed by incubation with Alexa 647 and 488 conjugated antibodies (Abcam Ltd., Cambridge, UK), respectively. After immunolabeling, the samples were mounted with ProLong^® Gold Antifade Mountant containing DAPI (Cell Signaling Technology, Danvers, MA, USA). For pseudovirus entry, the infected cells were obtained following the method from 4.4. Zsgreen was introduced to the pseudovirus beforehand, while the F-actin component of the cell membrane was stained with fluorescent phalloidin. All of the images were visualized and obtained utilizing a confocal inverted laser microscope (Leica SP8) with 48× magnification. The emission wavelength was 405 nm for DAPI, 488 nm for Zsgreen, and 505 nm for F-actin/phalloidin.