Nad nadh glo

The 3T3-L1 cells were seeded, differentiated, and treated with or without AZD3965 (1 µM) for 15 min, 30 min, 1 h, 4 h, 8 h, or 24 h in clear 96-well plates (Corning^®, Corning, NY, USA). Following incubation with the compound, the NAD⁺/NADH-Glo™ (Promega^®, Madison, WI, USA) kit was used to measure both the NAD⁺ and NADH concentrations within each sample according to the manufacturer’s protocol. Briefly, samples were lysed and split into 2 separate, white, 96-well plates (Corning^®, Corning, NY, USA). One plate was treated with a strong acid (0.4 N HCl) to eliminate NADH in the sample, while the other plate was treated with a strong base (0.2 N NaOH) to eliminate NAD⁺, and both plates were incubated at 60 °C for 15 min. Samples were then neutralized and subjected to the NAD⁺/NADH-Glo™ assay. Within this assay, in the presence of NAD⁺ or NADH, the enzyme ‘reductase’ reduced a proluciferin reductase substrate to form luciferin, which emitted a luminescent signal directly proportional to the quantity of NAD⁺ or NADH within the sample. Luminescence was read on a spectrophotometer (FlexStation 3; Molecular Devices, San Jose, CA, USA). To calculate the NAD⁺/NADH ratio for each sample, luminescence from acid-treated samples (reflecting NAD⁺ level) was divided by the luminescence signal from the corresponding base-treated samples (reflecting NADH levels).

Cells were cultured (1 × 10⁴ cells/well) 24 h in advance in 96-well white-walled clear-bottom tissue culture plates (Sigma) and treated with β-lap with or without DIC for 2 h. Then, ATP (CellTiter-Glo), hydrogen peroxide (H₂O₂) (ROS-Glo), and NAD/NADH (NAD/NADH-Glo) were assayed at the indicated time points after treatments using specific assays (Promega).