One glo

All experiments were performed using a robotic pipetting workstation (Hamilton Robotics Star Line) for high seeding, stimulation and revelation homogeneity. For each experiment, cells were seeded at 25000 cells/well in 96 well-plates and incubated 24 h prior to activation. Homogeneous cellular activations were performed using sodium butyrate (NaBut, 2 mM, Sigma) for PPARγ and ANGPTL4 reporter systems or 0,45 µm-filtered, sonicated E. coli (Epi300 from Epicentre, 10% vol/vol) for NFκB-SEAP system. Cells were then incubated 24 h prior quantification of the reporter gene expression (alkaline phosphatase (SEAP) or luciferase). SEAP (for NFκB system) secretion or intracellular luciferase expression (for both ANGPTL4 and PPARγ systems) were revealed using the QUANTI-Blue (Invivogen) or One-Glo (Promega) reagent respectively, according to the manufacturers’ instructions. All measurements were performed using a microplate reader (Infinite 200, Tecan) and quantified at 655 nm OD for SEAP and by luminescence for luciferase.

To determine infectivity, Nalm^+/c cells were infected with equal amounts of viruses normalized for p24 antigen and washed 3 times 6 hours post infection. Virus replication was monitored by quantifying p24 level in the supernatant on successive days using p24 ELISA (Innogenetics, Ghent, Belgium).
To determine the EC₅₀ in TZM-bl cells, 2x10⁴ cells were infected with equivalent amounts of virions produced in presence of a CX05045 1:3 dilution series. Cells were lysed in buffer containing 50 mM Tris/HCl, pH 7.3, 200 mM NaCl, 0.2% NP40 and 5% glycerol and analyzed for firefly luciferase activity (ONE-Glo™, Promega, Belgium) according to the manufacturer’s instructions. Readouts were normalized for protein content as determined by a BCA-assay (BCA Protein Assay Kit, Thermo Scientific).

20,000 HeLaP4 cells were seeded into 96-well plates on the day prior to infection. Cells were infected in triplicate with 3 dilutions (typically 1 × 10⁵, 3.3 × 10⁴ and 1.1 × 10⁴ pg p24) of VSV-G pseudotyped single-round HIV-1 supplemented with one of the compounds in a total volume of 200 µl per well. The virus was produced as described previously [31 (link)]. The final compound concentration in the assay was 100 µM. p24 measurements were performed with the Innotest HIV Antigen mAb kit (Fujirebio). 24 h after infection the supernatant was replaced by fresh medium. 72 h post infection cells were lysed in 50 µl of lysis buffer (50 mM Tris/HCl, pH 7.3, 200 mM NaCl, 0.2% NP40, 5% glycerol) and analyzed for firefly luciferase activity (ONE-Glo™, Promega, Belgium) according to the manufacturer’s protocol. Chemiluminescence was measured with a Glomax luminometer (Promega, Belgium). The signals were normalized for protein content as determined by a BCA protein assay (Thermo Scientific Pierce).

NAb titers against SARS-CoV-2 and VOC spike in participant-derived polyclonal IgG fractions were assessed by previously developed HIV neutralization assays [22 (link)], subject to minor modifications. Briefly, participant-derived IgG was 3-fold serially diluted on a 96-well plate from starting concentrations of 100 µg/mL or 300 µg/mL. Pseudoviruses were thawed to 37 °C and added at a concentration of 3 ng/well of the proviral backbone p24 antigen level and incubated at 37 °C and 5% CO₂. One hour after incubation, freshly trypsinized target cells (293T/ACE2/TMPRSS2) were added to the IgG/pseudovirus complex at a density of 22,000 cells/well and incubated for 48 h. A luciferase substrate (ONE-Glo, Promega, cat # E6130, Madison, MI, USA) equipped with a lysis agent was added to the culture supernatant in accordance with the manufacturer’s instructions. IgG neutralization at each dilution was calculated as a percentage reduction in luminescence in the IgG + pseudovirus wells relative to the pseudovirus-only wells. IgG NAb titers were expressed as IC₅₀ values calculated on a dose–response curve fit with a non-linear function. An IgG sample of known NAb titer was used as a positive control to ensure consistency in conditions between assay runs.

One million HEK293 cells (Promega) were plated on a T25 dish. After 8 h, cells were transfected with 5 μg of a GAS reporter vector (pGL4[luc2P/GAS-RE/Hygro], Promega) and 15 μl FuGENE HD (Promega, cat no. E2311) according to the manufacturer’s instructions. 16 hours post-transfection, cells were plated on pre-plated siRNAs as described above. Cells were incubated on siRNAs for 48 hours before activation with 100 ng/ml IFNγ (Peprotech) after which incubation was continued for 24 hours. Subsequently, cell viability and reporter activity were measured with CellTiter-Fluor and ONE-Glo reagents (Promega), respectively.