μ slide 8 well chamber slide well

HEK293T cells were seeded in a μ-Slide 8-Well Chamber Slide-well (iBidi GmbH, Martinsried, Germany) after precoating with poly-L-Lysine (Cultrex, R&D Systems, Minneapolis, MN, USA). After overnight culture, the cells were transfected with the plasmids encoding WT hTIM-1 or its SNV mutant genes. At 24 h post-transfection, the cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. After washing with PBS, the cells were incubated with PBS containing 3% bovine serum albumin for blocking for 1 h at room temperature. After washing 3 times with PBST, the cells were incubated with a goat anti-hTIM-1 polyclonal antibody (AF1750, R&D Systems, Minneapolis, MN, USA) as the primary antibody for 1 h at room temperature. The cells were washed 3 times with PBST and then incubated with a donkey anti-goat IgG polyclonal antibody conjugated with fluorescein isothiocyanate (FITC) (sc2024, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a secondary antibody, followed by counterstaining with 1 μg/mL 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (D1306, Molecular Probes, Eugene, OR, USA) for 1 h in the dark at 4 °C. Images were acquired with a 63 × oil objective lens on a Zeiss LSM700 inverted microscope using ZEN 2009 software (Carl Zeiss, Oberkochen, Germany).

Immunofluorescence experiments were performed using μ-Slide 8 Well Chamber Slide-well (iBidi GmbH). Caco-2 cells or 293T-hACE2 cells were infected with SARS-CoV-2 virus produced in 293T-hACE2 or 293T-hACE2-ΔFURIN. In order to add approximately the same number of viral particles for both conditions, normalisation was performed by quantifying the SARS-CoV-2 nucleoprotein band in the Western blot (Fig 4D) using ImageJ software. Cells were fixed with 4% formaldehyde after 24 hours post infection, permeabilised with 0.1% Triton X-100 and then blocked with 5% FBS. Primary antibody used was mouse anti-spike (GeneTech, clone 1A9) and secondary was anti-Mouse Alexa Fluor 488. Nuclei were stained with Hoechst 34222. Images were taken with Zeiss 710 confocal microscope. Image processing was performed using ImageJ software.