SARS-CoV-2 S Protein Cell-Cell Fusion Assay

The DSP assay was performed as described previously (24 (link)). Briefly, effector cells expressing S protein and target cells expressing CD26 or ACE2 alone or together with TMPRSS2 were seeded in 10-cm plates and incubated overnight (Fig. S1a and b). Cells were treated with 6 μM EnduRen (Promega), a substrate for Renilla luciferase (RL), for 2 h. To test the effect of inhibitor, 0.25 μL of compound library or 1 μL of selected inhibitor dissolved in DMSO was added to the 384-well plates (Greiner Bioscience, Frickenhausen, Germany). Next, 50 μL of each single-cell suspension (effector and target cells) was added to the 384-well plates by using a Multidrop dispenser (Thermo Fisher Scientific). After incubation at 37°C in 5% CO₂ for 4 h, the RL activity was measured using a Centro xS960 luminometer (Berthold, Bad Wildbad, Germany).