Luminescent assays

Total ATP and NAD levels were evaluated using luminescent assays (Promega, Madison, WI, USA) according to the manufacturer’s protocol. In brief, WSU-DLCL2 and WSU-FSCCL cells were grown in opaque walled 96-well plates and exposed to increasing KPT-9274 concentrations for indicated periods. ATP levels were determined by adding CellTiter-Glo Luminescent cell viability reagent (catalog number G7571) to each well and incubated for 30 min. NAD/NADH levels were obtained by adding NAD/NADH-Glo reagent (catalog number G9072) and incubated for 30 min. Luminescent signal was then detected using SpectraMax i3x and SoftmaxPro software.

For lactate, LDH activity, NAD+/NADH and NADP+/NADPH measurements, cells were harvested at various time points following drug treatment using appropriate buffers and frozen in liquid nitrogen. Intra-cellular lactate, NAD+, NADH, NADP+ and NADPH levels along with ex vivo LDH activity were analyzed using commercially available colorimetric assays (BioVision, CA, USA), according to the manufacturer’s instructions as previously described by our group and others^{17 ,19} . Intra-cellular ATP levels were measured using commercially available luminescent assays (Promega, WI, USA) as previously described by our group and others; ATP levels were normalized to cell number quantified using Hoechst staining as previously described by our group^{18 (link)}.