White clear bottom 96 well plate

Intracellular pyruvate was measured using the Amplite™ Fluorimetric Pyruvate Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s instructions. The 3T3-L1 cells were seeded and differentiated in white, clear-bottom, 96-well plates (Corning^®, Corning, NY, USA). After differentiation, cells were treated with compound or vehicle as indicated. Following 15 min, 30 min, 1 h, 4 h, 8 h, or 24 h incubation with 1 µM AZD6965, media was removed and cells were lysed with ReadiUse™ mammalian cell lysis buffer (AAT Bioquest, Sunnyvale, CA, USA). Lysates were then subjected to an enzyme-coupled reaction with the Quest Fluor™ Pyruvate Sensor, resulting in an absorbance signal detected at 575 nm which was proportional to the quantity of pyruvate within the sample. Absorbance was read on a spectrophotometer (FlexStation 3; Molecular Devices, San Jose, CA, USA).

Measurements of the intracellular ROS levels were performed using the DCFDA/H2DCFDA cellular ROS assay kit (Abcam, Amsterdam, Netherlands) according to the manufacturer’s protocol. In brief, cells were seeded on white clear-bottom 96-well plate (Corning, Corning, USA) and incubated overnight. Cells were treated with 10 mM APAP, 2 mM H₂O₂, or 300 µM TBHP (H₂O₂ and TBHP served as positive controls for ROS induction) for 6 hr in FCS-free cell culture medium. Fluorescence was measured using a microplate reader (FluoStar Omega, BMG Labtech GmbH, Ortenberg, Germany). Buffer solution without cells served as a background control.

Nrf2/ARE-luciferase
reporter stable HEK293 cell line was purchased from Signosis (Santa
Clara, CA). The cell line has been stably transfected with the pTA-ARE-luciferase
reporter vector, which contains four repeats of antioxidant response
elements (ARE) of the promoter upstream of the firefly luciferase
coding region along with a hygromycin-resistant vector. Cells were
cultured and tested according to the manufacturer’s manual.
Briefly, the stable reporter cell line was cultured in DMEM culture
medium containing 10% FBS, 1% PNS, and 50 μg/mL hygromycin B
(Sigma-Aldrich, St. Louis, MO) in humidified air with 5% CO₂ at 37 °C. When reached 90% confluency, cells were trypsinized
and seeded onto a white clear-bottom 96-well plate (Corning, Corning,
MA) at a density of 2 × 10⁴ cells/100 μL/well
and cultured overnight. Cells were treated with various conditions
for the designated time, washed with PBS before lysing with lysis
buffer (Signosis), and incubated with luciferase substrate solution
(Signosis). The chemiluminescent signal was read by a PerkinElmer
Vector 3 plate reader (Waltham, MA). After subtraction with the signal
of the blank wells, the chemiluminescence signal of all treatment
groups was normalized by the signal of the nontreatment group (naive
reporter cells).

For Nluc readouts, 1.2 × 10⁴ A549-hACE2 cells per well were suspended in 50 μL infection medium, seeded into a white clear-bottom 96-well plate (Corning), and incubated overnight at 37°C with 5% CO₂. On the following day, compounds were added directly to cultures as 3-fold serial dilutions with a Tecan D300e digital liquid dispenser, with DMSO volumes normalized to that of the highest compound concentration (final DMSO concentration of <0.1%). SARS-CoV-2-Nluc viruses were diluted to an MOI of 0.05 and aliquoted at 50 μL/well. At 48 hpi, 75 μL Nluc substrate solution (Promega) was added to each well. Luciferase signals were measured using an Envision microplate reader (Perkin Elmer).
For firefly luciferase readouts, the assay setup was the same as for the Nluc assay, except cells were infected with SARS-CoV-2-Fluc viruses at an MOI of 1.0, and at 48 hpi, 100 μL One-Glo luciferase substrate solution (Promega) was added to each well prior to reading the signal on the Envision plate reader (Perkin Elmer).