Flexstation 3

HEK293T cells were obtained from the Center of Animal Science and Animal Medicine, Shandong Agricultural University. The cells were cultured at 37 °C in DMEM (Thermo Fisher Scientific, USA) containing 10% FBS (BioInd, Israel) and 1% P/S (Thermo Fisher Scientific, USA). Before transfection, the cells were seeded onto 24-well plates. When the concentration reached 1 × 10⁵ per well, the DMEM was removed, and Opti-MEM was added to incubate the cells. Then, 500 ng recombinant constructs and 50 ng pRL-TK were cotransfected into the cells in 400 µl opti-MEM medium using Lipofectamine TM 3000 (Invitrogen, USA) according the manufacturer’s instructions and incubated at 37 °C for 6 h. After that, opti-MEM medium was removed, and DMEM was incubated for 48 h. Cells were collected, and luciferase activity assays were performed using a dual-luciferase kit (Promega, USA) on a Flexstation 3.

The qualification of NADH and NAD⁺ was performed by using the NAD/NADH-Glo Assay kit (Promega) and FlexStation 3 following the manufacturer’s protocols. The samples (100 µL) at an OD₆₀₀ of 0.4 to 0.6 were mixed with an equal volume of NAD/NADH-Glo detection reagent in 96-well opaque plates and incubated at room temperature with gentle shaking for 60 minutes. RLU values were recorded by FlexStation 3 and normalized by OD₆₀₀.