Developer toolbox

To evaluate the effect of the tested compounds on cell viability, in the absence of viral infection, Vero CCL-81 cells were seeded 24 h prior to compound addition in 96-well imaging plates, at a density of 1 × 10⁴ per well, and cultured overnight at 37 °C in 5% CO₂. Five final concentrations (10.00 μM, 14.96 μM, 22.36 μM, 33.44 μM, and 50.00 μM) of each compound resuspended in DMEM containing 5% FBS were tested in the seeded cells (in duplicate), by replacing the culture medium. DMSO was used as control. After 48 h incubation with the compounds, cells were fixed, permeabilized, and stained for 30 min for nuclear markers, with DAPI (Thermo Fisher, Waltham, MA, USA), and for cell delineation markers, with HCS CellMask (Thermo Fisher, Waltham, MA, USA). In the end, images of each well were acquired on the IN Cell Analyzer 2000, using a 10× magnifying objective. Image analysis was performed by using image segmentation and the quantification software “Developer Toolbox” (version 1.9.3, x64, GE Healthcare, Chicago, IL, USA) and CellProfiler [35 (link)]. DAPI was used to assess the number of cells. Three independent experiments were performed. Data were presented as percent compared with control wells (“blank”, non-infected cells without the presence of the compounds). The IC₅₀ values in the absence of infection were calculated with Prism 9 software.

To measure total green fluorescence signal per worm, a young adult-stage population was washed from the plate and transferred to clear, flat bottom, 96-well plate. Worms were paralyzed with 10 mM levamisole (L9756, SIGMA-ALDRICH). Plates were sealed with a seal plate adhesive (SealMate System, Excel Scientific) and imaged in the automated microscope IN Cell Analyzer 2000 (GE Healthcare Life Science) using an objective Plan Apo 2X/0.1. Total GFP intensity was calculated with the Developer Toolbox (GE Healthcare Life Science) integrated software.