Fig. 1

FK866 affects NAD⁺(H) and ATP levels in Jurkat cells leading to cell death. a Flow-cytometric quantification of cell viability with AnnexinV (FITC) and 7AAD (PerCP-Cy5-5-A) staining. Jurkat cells were treated with FK866 for 48,72 and 120 h. Mock, 5 nM FK866 and 100 nM FK866 (at 48 and 72 h) are shown as representative samples. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on the acquisition of 10000 events/sample. b Cell-cycle analysis with PI staining of the nuclei after 48 h of treatment. Overnight serum starvation is shown as positive control of induced cell cycle synchronization in G0/G1 phase. Histograms quantify the cell cycle phase distribution. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on acquisition of 30000 events/sample. Cell cycle phase analysis was done using ModFit LT 3.2 software and the Sync Wizard model. c Jurkat cells were treated with FK866 for 48 h. Alternatively, cells were exposed to 5 μM of Camptothecin for 4 h as a positive control. Protease activity in cell extracts was assessed with a commercially available kit and values were normalized to the protein concentration in the same extracts. d Caspase 3/7 activity was measured in Jurkat cells treated as in c. e Jurkat cells were treated with FK866 for 48 h. Thereafter, intracellular NAD⁺(H) and ATP levels were evaluated in comparison with control Jurkat cells. RLUs were normalized to number of viable cells. In c-e the means with SD of at least three independent experiments are shown. Statistical significance was calculated with t-test (* and # indicates p-value <0.05)