Figure 6.

IFITM overexpression inhibits ACE2 cell surface expression

(A) Proximity ligation assay between ACE2 and IFITM1, IFITM2 or IFITM3 in transfected HeLa cells. Scale bar, 20 μm. Bars represent the mean of two independent experiments (±SEM), ∗∗∗∗p < 0.0001.

(B) HeLa-ACE2 cells were transfected with IFITM1, IFITM2 or IFITM3 expression constructs or an empty control vector. Forty-eight hours post-transfection cells were stained with anti-ACE2 and anti-IFITMs antibodies and examined by confocal microscopy. Scale bar, 20 μm. ACE2 signal intensities at the cell surface and in the cytoplasm were quantified using ImageJ. ∗∗∗∗p < 0.0001.

(C) Intensity of ACE2 staining throughout the cell in the absence and presence of IFITM overexpression quantified using ImageJ.

(D and E) TZM-bl cells (D) or A549 stably expressing TMPRSS2 and ACE2 (E) were transfected with either IFITM1, IFITM2, or IFITM3 expression constructs. 48 h post-transfection, cells were permeabilized (left panels) or not (right panels), stained with anti-CD4 or anti-CD46 antibody respectively and analyzed by flow cytometry. Shown are mean fluorescence intensities (MFIs) measured in cells transfected with IFITM expression vectors relative to those that received the control construct (100%). Bars represent the mean of four independent experiments (±SEM), ∗∗, p < 0.01.

(F) A549 stably expressing TMPRSS2 and ACE2 cells were transfected with flag-tagged IFITM1, IFITM2 or IFITM3 expression constructs or an empty control vector. Forty-eight hours post-transfection cells were stained with anti-CD46 and anti-flag antibodies and examined by confocal microscopy. See also Figure S3.