Extended Data Fig. 2: Development of assays to investigate the mechanism of cGAMP export.

a, Schematic of experiment for b. CD14⁺ PMBCs were stimulated with increasing concentrations of extracellular cGAMP for 16 h. b, IFNB1 mRNA levels were normalized to indicated gene and fold induction was calculated relative to untreated CD14⁺ cells. Each donor was performed as one independent experiment; 2 qPCR replicates are plotted with a bar representing the mean. c, Coomassie gel of recombinant mouse ENPP1 purified from media; elution fractions were pooled before use (left). ³²P-cGAMP degradation by mouse ENPP1 analyzed by TLC (right). Data are representative of 10 independent experiments. d–e, 293T cGAS ENPP1^low cells were incubated with serum-free ATP depletion media (no glucose, 6 mM 2-deoxy-D-glucose, 5 mM NaN₃) or serum-free complete media for 1 hour. Levels of analytes were measured: (d) ATP by LC-MS/MS, total protein by BCA, cell death by extracellular lactate dehydrogenase activity, and (e) cGAMP by LC-MS/MS. BQL = below quantification limit. Data are from 1 experiment; 3 cell culture replicates are plotted (except for extracellular cGAMP, where 2 cell culture replicates are plotted). ATP data are representative of 3 independent validations (shown in Supplementary Fig. 5).

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