Measurement of NAD+ levels

Cellular NAD⁺ levels were assayed using the NAD⁺/NADH Quantification Colorimetric Kit (BioVision) according to the manufacturer’s protocol. Briefly, U2OS cells were pre-treated with DMSO, 25 μM PARGi or 10 nM FK866 for 4 days. Cells were trypsinized, washed with ice-cold PBS and counted. 20,000 cells per sample were resuspended in NAD⁺/NADH Extraction Buffer and lysed by two repealed freeze thaw cycles on dry ice. Cells were vortexed and centrifuged at 14,000 rpm for 5 min to pellet cell debris. The supernatant was passed through a 10 kDa Spin Column (Abcam) at 10,000 g for 15 min to remove cellular enzymes that utilize NAD⁺/NADH as coenzymes. The supernatant from each condition was split into two to allow separate measurements of: 1) NADH-only and 2) NADt (NAD⁺ & NADH combined). To measure NADH-only, NAD⁺ was depleted by heating the samples to 60°C for 30 min. 100 μL of each sample or standard was placed in a white 96-well flat clear bottom plate (Corning). 100 μL NAD⁺ Cycling Enzyme Mix was added to each well before mixing on a plate shaker for 5 min. 10 μL of NADH Developer Solution was added to each well before mixing using a plate shaker for 15 min. The plate was incubated at room temperature for 30 min before measuring the absorbance 450 nm. NADH-only and NADt levels were calculated from absorbance values using a standard curve. NAD⁺ measurements were calculated by subtracting NADH-only values from NADt values. NAD₊ measurements were normalized to the total protein content of each sample. Total protein content was determined using Bradford Protein Assay (Bio-Rad) according to the manufacturer’s protocol.