Fig. 3: Analysis of cell death in ADA2^-/- U-937 cells at baseline and after induction of apoptosis and necroptosis in vitro.

A Cell death in U-937 cells was measured by flow cytometric measurement after staining with Annexin V and Zombie® viability dye. Cells were incubated for 5 h. Apoptosis and necroptosis were induced by stimulation with 100 nM birinapant (SM) ± 20 µM Z-VAD-FMK (Z-VAD) followed by 20 ng/mL TNF-α. Inhibition of necroptosis was achieved by adding 10 µM necrostatin-1s (Nec-1s). The experiment was performed for n = 5 distinct ADA2^-/- U-937 cell clones. B Induction of apoptosis and necroptosis in U-937 cells by western blot. Whole cell lysates were produced after 5 h incubation as for (A). The plots show protein levels from n = 5 independent experiments. All blots are depicted in Supplementary Fig. S2C. C Cell death in lymphocytes from healthy controls (HC) and DADA2 patients was measured by flow cytometry after staining with Annexin V and Zombie® viability dye after 48-hour incubation in vitro. Data from n = 9 (HC) and n = 8 (DADA2, comprising n = 7 distinct patients) independent experiments are shown. The median is indicated in all plots. Mann-Whitney-U test, *p < 0.05. The patients’ phenotype is indicated by colour as displayed in Fig. 1B. Legend: CC3 cleaved caspase-3, MLKL mixed lineage kinase domain like pseudokinase, Nec-1s necrostatin-1s, SM Smac mimetic, Z-VAD Z-VAD-FMK.