Fluorescence microscopy

Nile red staining was applied for evaluating lipid accumulation in HepG2 cells with different treatments. After incubating with PA and/or AEPL for 24 h, the medium was removed and cells were fixed with 10% paraformaldehyde for 10 min at room temperature. HepG2 cells were stained with 2 μg/mL Nile red for 15 min. Images were acquired by fluorescence microscopy (Bio-Rad, Hercules, CA, USA) and the area was quantified by ImageJ software.

NRK−52E cells were seeded in a 6−well dish (2x10⁵ cells/dish) for 24 h. NRK−52E cells were treated with or without LSE (5 and 10 μg/mL) for 24 h in the presence or absence of cisplatin (8 μM). After 24 h, the medium was removed and 4% formalin was added to fix for 30 min. Next, DAPI (Sigma-Aldrich, St. Louis, MO, USA) was diluted with PBS and the nucleus was stained for 20 min at room temperature in the dark. Cell morphology was captured by fluorescence microscopy (Bio-Rad, Hercules, CA, USA) and quantified by ImageJ.

Cell–cell fusion assays were conducted as previously described [9 (link), 29 (link)]. Briefly, HEK293T cells transiently transfected with hACE2 and TMPRSS2 were used as target cells, and HEK293T cells transiently transfected with SARS-CoV-2 S protein and GFP were used as effector cells. 40 h after transfection, target cells were treated with vehicle, TAT or peptide (10 µM) 15 min before adding the effector cells. The effector cells were detached from their culture dishes with 0.25% trypsin and overlaid onto a target cell monolayer at an effector:target cells ratio of 1:3. After 2 h incubation, 10–21 images were randomly taken to count the number of fused vs. unfused cells using fluorescence microscopy (Bio-Rad). The cells number were counted using ImageJ (NIH). Effector cells incubated in DMEM supplemented with 10% FBS without target cells were used as a negative control. All experiments were performed in triplicate. This assay was performed in a double-blind fashion.