BafA1 and Niclosamide affect RBD uptake in HEK-293T cells and reduce Spike pseudovirus infection in HEK-293T and A549-ACE2 cells.

A-D: HEK-293T cells were treated with Control (0.4%DMSO), BafA1 400nM or Niclosamide 10μM for 30 minutes and pulsed with RBD and transferrin (A and B) or RBD and dextran (C and D) for 30 minutes with or without inhibitors. Images are shown in A and C, quantification is shown in B and D. RBD and dextran uptake is robustly reduced, while transferrin uptake increases upon treatment with BafA1 and Niclosamide. Number of cells ≥ 75 for each treatment. p-value table is indicated in S1 Table. E: Images show Spike-pseudovirus transduced mCherry positive HEK-293T cells in the presence of NH₄Cl 20mM, CQ 10μM, BafA1 50nM and Niclosamide 5μM. F: Quantification of the normalized area of mCherry positive cells as a proxy for transduction, shows a reduction in transduction efficiency upon treatment with NH₄Cl 20mM and CQ 10μM compared to 0% DMSO (Control₁), 25nM and 50nM of BafA1 compared with 0.05%DMSO (Control₂), 1μM and 5μM of Niclosamide compared to 0.1%DMSO (Control₃). Number of repeats = 3 for each condition except 4 for 0%DMSO. The data is represented as mean +/- SD. G: Toxicity, as assessed by MTT based colorimetric assay, is represented as percentage viability of cells upon treatment with NH₄Cl 20mM and CQ 10μM compared to 0% DMSO (Control₁), 25nM and 50nM of BafA1 compared with 0.05%DMSO (Control2), 1μM and 5μM of Niclosamide compared to 0.1%DMSO (Control3). Number of repeats = 3 for each condition except 9 for 0%DMSO. The data is represented as mean +/- SD. H-I: Quantification of the mCherry positive cells in A549-ACE2 cells, as a proxy for transduction, shows a reduction in transduction efficiency upon treatment with 50nM of BafA1 in H and range of concentrations of Niclosamide in I (compared to respective DMSO controls). Data representation in B, D are as described in Fig 1 and H, I as in Fig 3. Scale bar: 40μm (A, C), 100μm (E).

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