Britelite reagent

HTS was performed as previously described [24 (link)]. Briefly, the HCT116 cells used expressed firefly luciferase under CMV-promoter control (HCT116/CMVpLUC cells) or under control of the S100A4-promoter (comprising the sequence from −1487 bp upstream- to the S100A4 transcription start site; HCT116/S100A4pLUC cells). The S100A4 promoter sequence was a kind gift from David Allard (Peninsula Medical School, University of Exeter and University of Plymouth, Exeter, UK). The HCT116 cells were transfected with the S100A4 cDNA, kindly provided by Claus Heizmann (University of Zurich, Zurich, Switzerland; HCT116/S100A4 cells) or the empty vector as the control (HCT116/vector cells). Stable transgene expressing cells were selected with 1 mg/mL neomycin (PAA Laboratories, Cölbe, Germany) or 1 µg/mL puromycin (Invitrogen, Carlsbad, CA, USA).
2.5 × 10³ cells/well of HCT116/S100A4pLUC cells were seeded in 384-well plates, and the cells were treated for 24 h with each compound of the LOPAC 1280 library. Luciferase expression was determined by Britelite reagent (Perkin Elmer, Waltham, MA, USA). In parallel, the cytotoxicity of the compounds was measured by the AlamarBlue™ cytotoxicity assay (AbD Serotec, Raleigh, NC, USA). Reporter inhibition efficacy was determined by the ratio of toxicity versus activity.

Virions pseudotyped with Ebola virus GP_Δmucin (EBOV GPΔ) and Lassa virus GP were produced in HEK293T/17 cells using Lipofectamine 2000 (Invitrogen). Cells grown to 60–80% confluency were transfected with 4.65-µg psPAX2 (Gag-Pol helper vector), 6.25-µg pFSW-Tat, 1-µg pHIV-Rev, and 1.5-µg of EBOV GP or LASV GP. Four to six hours post-transfection, the media were replaced with phenol red free DMEM supplemented with 10% FBS, 2-mM L-glutamine, and 1-mM sodium pyruvate. Pseudovirus-containing media were collected 48-h post-transfection and clarified by low-speed centrifugation. HIV pseudoviruses were pelleted through a 25% sucrose-HME cushion and resuspended in HME buffer without sucrose. The pseudovirus preparation was aliquoted and stored at −80°C. The concentration of HIV p24 was measured by enzyme linked immunosorbent assay (ELISA) (82 (link)).
Infection of TZM-bl cells by HIV pseudoviruses was performed as described by Sarzotti-Kelsoe et al. (83 (link)). Firefly luciferase activity was measured 2 days post-infection with BriteLite reagent (PerkinElmer Life Sciences) in a plate reader (GloMax Explorer, Promega). HIV pseudovirus preparations were diluted in Opti-MEM (Gibco) to the same concentration of p24.

Production of HIV-1 based single round luciferase reporter virus (HIV-luc) was performed by transfection of HEK293T cells with an HIV-1 based luciferase reporter virus defective in env (pNL43lucE-R+66 (link)), and pseudotyped with VSV-G coding expression plasmid67 (link) in a ratio of 2:1 using Lipofectamine2000 according to the manufacturer’s instructions (Invitrogen/Life Technologies). A third generation lentiviral single round luciferase reporter vector (lenti-luc) was produced as previously described (CSII-luc68 (link)). Viral particles were harvested 48 and 72 hours post transfection, filtered with 0.45 μm pore size sterile filter and stored at −80 °C. For transduction, reporter viruses were added to cells and spinoculated at 290 g at 30 °C for 90 minutes. Luciferase activity was measured 24 hours after transduction using BriteLite reagent according to manufacturer’s protocol (PerkinElmer). Chemiluminescence, measured in relative light units (RLU), was detected using a PherastarFS plate reader (BMG).

An NF-κB reporter (Luc)—HEK293 cell line was generated by lentiviral infection using the Cignal Lenti NF-κB Reporter Luc system (SA Biosciences CLS-013L-8,) and was cultured in DMEM/GlutaMAX™ (Gibco) containing 4.5 g/L D-Glucose and 25 mM HEPES, supplemented with 10% heat-inactivated fetal calf serum (PAA). For cell passaging, the complete medium was supplemented with 1 μg/ml Puromycin (Sigma). One day before the experiment, the medium was changed for fresh medium without antibiotic. Transfection was performed in sterile Costar Corning^® 96 well white flat bottom polystyrene TC-treated microplates using cell suspensions of 30’000 cells/ well using 0.1 μg total plasmid DNA and 0.3 μl X-tremeGENE™ 9 DNA transfection reagent (Roche). Transfected cells were incubated overnight at 37°C before lysis into Britelite reagent (PerkinElmer^®). Luciferase activity was analyzed using an EnVision Multilabel Reader.