Steady glo luciferase assay reagent

Inhibition of HCV replication was measured in huh7 cells harboring HCV replicon (GT 1b-Con1 strain)-containing luciferase (luc-ubi-neo/ET) reporter gene according to the previously published protocol^{35 (link)}. The luciferase reporter in this assay is an indirect measure of HCV replication, as HCV replication is proportional to the luciferase activity. Sub-confluent Luc-ubi-neo/ET cells plated in 96-well plates in triplicates were treated with various concentrations of the compounds and incubated at 37 °C with 5% CO₂. Luciferase activity (as a measure of HCV replication) was measured 72-hours later, using Steady-Glo Luciferase assay reagent (Promega). Each run also included two positive controls-the NS5B inhibitor Sofosbuvir, and recombinant human interferon alpha-2b (rIFNα-2b). Cell viability was measured by CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega) in parallel to determine CC50. Selectivity index 50% (SI50) were calculated by the ratio of CC50/EC50 and CC50/EC90 as mentioned above for HBV assay.

The TZMbl cells were seeded in the wells of a 96-well plate (10,000 cells/well) with complete DMEM. On the next day, medium was removed from each well and cells were washed once with 200 µl incomplete DMEM. Cells were starved for 1 hr in 100 µl incomplete DMEM, which was then removed and different dilutions of virus were added in a total volume of 100 μl of incomplete DMEM. After 4–6 hr of incubation at 37°C in a 5% CO₂ incubator, the virus containing medium was removed and 100 µl of complete medium was added. After 48 hr of incubation, the luciferase reading was taken using Steady-Glo luciferase assay reagent according to manufacturer's instructions (Promega, Madison, USA).

Monolayers of HEPG2 cells established in 6-well format were infected with log-phase C. albicans (CaCi-2) at a concentration of 2.5 × 10³ cells/mL in DMEM supplemented with 5% fetal bovine serum and pen-strep antibiotic. After addition of test compounds, culture was continued for 48 h. Wells were fixed in 10% formalin, stained with a periodic acid-Schiff reagent kit (Sigma, 395B-1KT) per manufacturer’s recommendation and photomicrographs obtained under standard white light illumination. To quantitate the concentration-dependent toxicity of Hsp90 inhibitors in human-fungal co-cultures, 293T cells stably expressing firefly luciferase were plated in 384-well format (2000 cells in 20 µL per well, black clear-bottom plates) and allowed to adhere overnight. Wells were then infected with log-phase GFP-marked CaCi-2 (CaLC867, 2.5 × 10³ cells/mL) in an equal volume of medium supplemented with fluconazole (4 µg/mL). After 48 h, medium was replaced with PBS and relative fluorescence per well measured on a plate reader (Tecan Infinite M1000Pro, 480 nm excitation and 540 nm emission). Steady-Glo luciferase assay reagent (10 µL per well, Promega Cat# E2520) was then added, plates incubated at room temperature for 10 mins and relative luminescence per well measured using an Envision plate reader (Perkin Elmer).

The cells were prepared in the same way as described for the bioluminescence rhythm assay. The cell spot on agar piece was flash frozen in liquid nitrogen in a 1.5-mL tube and stored at -80°C. The cell spots were lysed by adding 150 μL of Steady-Glo Luciferase Assay Reagent (Promega) and vortexed for 10 min. After the agar piece settled down, 80 μL of the supernatant was used for monitoring the luminescence in a multilabel plate reader (ARVO X4, PerkinElmer).

A SARS-CoV-2 pseudovirus, incorporating a luciferase reporter gene, was generated similar to previously described approaches^{1 (link),2 (link),21} . To generate pseudoviruses, HEK293T cells were co-transfected with packaging construct psPAX2 (AIDS Resource and Reagent Program), a luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and a plasmid expressing S protein, pcDNA3.1-SARS-CoV-2 SΔCT with lipofectamine 2000. 48 h after transfection, supernatants containing pseudoviruses were collected and filtered through a 0.45-µm filter. To quantify neutralizing activity of human plasma samples or purified IgG stocks, HEK293T target cells, transfected to express human ACE2 (HEK293T-hACE2), were seeded in 96-well tissue culture plates at a density of 1.75 × 10⁴ cells per well and cultured overnight. Three-fold serial dilutions of heat-inactivated plasma or purified IgG samples were prepared, mixed with 50 µl of pseudovirus, incubated for 1 h at 37 °C, and then added to HEK293T-hACE2 seeded wells. After 48 h of incubation, cells were lysed in Steady-Glo Luciferase Assay Reagent (Promega) according to the manufacturer’s instructions. SARS-CoV-2 neutralization titers were defined as the sample dilution at which a 50% reduction in relative light units was observed relative to the average of the control wells treated with virus only.

For the ARE-luciferase reporter assay, HEK293-ARE reporter cells were seeded into 96-well plates at a density of 5 × 10⁴ cells/mL and treated as indicated. Luciferase activity was detected using Steady-Glo^® Luciferase Assay Reagent (E2520, Promega) according to the manufacturer’s instructions.
For the dual-luciferase reporter assay, RAW264.7 cells were transfected with pGL4.13-Nfkbiz-promoter or pGL4.13-Nfkbiz-3UTR together with pRL-TK as the internal control for 24 h. Cells were then treated as indicated and harvested. The dual-luciferase activity was determined using the Duo-Lite Luciferase Assay Reagent (DD1205-01, Vazyme).