Half area 96 well plate

The tubulin polymerization assay was performed by Ecrins Therapeutics Services (www.ecrins-therapeutics-services.com). Briefly, a half-area 96-well plate (Greiner Bio-one, Courtaboeuf, France) was charged with pure tubulin (40 µmol.L⁻¹) in a PIPES-based polymerization buffer (80 mM K-PIPES pH 6.8, 1 mM MgCl₂, 1 mM EGTA). After the addition of 1 mmol.L⁻¹ GTP, tubulin assembly was followed at 37 °C in the presence of colchicine, paclitaxel or increasing concentrations of PP-13, by turbidimetry variation at 350 nm every 30sec during 1h (FLUOstar Omega, BMG Labtechnologies). The experiment was performed in triplicate. A negative control contained an equivalent quantity of DMSO as the sample with higher concentration of PP-13 (25 µmol.L⁻¹). The solubility of the compound in the polymerization buffer was checked prior to the test.

For mAb neutralization assays against PDCoV S VSV, HEK293T cells were transfected with hAPN as described in the cell entry assay. 40,000 cells/well were seeded into 96-well plates coated with poly-lysine and incubated overnight at 37°C. Antibody titration series was performed in a half-area 96-well plate (Greiner) starting at a 2x concentration of 20 μg/ml and diluting 1:3 with DMEM. Equal volume of pseudovirus was added and incubated at room temperature for 30 minutes and 40 μL was transferred to cells and incubated for 2 hours at 37 °C with 5% CO₂. Cells were supplemented with 40 μL of DMEM with 20% FBS and 2% PenStrep and incubated for 22 hours at 37 °C with 5% CO₂. After 22 hrs, 40 μL of One-Glo-EX substrate (Promega) was added to each well and incubated on a plate shaker in the dark for 5 min before reading the relative luciferase units using a BioTek Neo2 plate reader. Relative luciferase units were used to determine (%) neutralization in Prism (GraphPad). Nonlinear regression curve fit and sigmoidal 4PL where x is the concentration was run to determine IC50 values for 3 biological replicates of pseudovirus and three technical replicates for each titration series.