RBD uptake is sensitive to acidification inhibitors.

A, B: AGS cells were treated with Control (0.3% DMSO, 0.6% DMSO, 0% DMSO) or inhibitors (BafA1 200nM, BafA1 400nM, NH₄Cl 30mM) for 30 minutes and then pulsed with RBD, transferrin and dextran for 30 minutes with or without inhibitors. Images are shown in A and quantification in B with total cell mean intensity shown in (i), the number of endosomes shown in (ii) and intensity per endosome shown in (iii) for each probe in each condition. Control₁ is 0.3% DMSO, Control₂ is 0.6% DMSO and Control₃ is 0% DMSO. Number of repeats ≥ 4 for each treatment and each repeat has >80 cells. C, D: AGS cells transfected with myc-ACE2 were treated with Control (0.2%DMSO) or BafA1 400nM or Niclosamide 10μM for 30 minutes and then pulsed with RBD for 30 minutes. The cell surface-bound RBD was stripped using ascorbate buffer and cell surface ACE2 was labelled using anti-myc antibody. Normalized RBD uptake is quantified as the ratio of the amount of internalized RBD to the amount of surface ACE2. Images depicted in C and quantification in D show that there is a reduction of RBD uptake upon treatment with BafA1 (p-value < e-08) or Niclosamide (p-value < e-07) in transfected as well as untransfected cells. Number of cells > 50 for each condition. Data representation in B and D are as described in Figs 2 and 1, respectively. Scale bar: 40μm (A, C).

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