Extended Data Figure 5. Covalent inhibition of SLC19A1 by NHS-methotrexate blocks STING activation.

a, Schematic overview of CDN-induced phosphorylation (P) of STING and downstream effectors TBK1 and IRF3. b, THP-1 monocytes pre-treated with DMSO or NHS-methotrexate (MTX) (5 μM) were treated with varying concentrations of 2’3’-cGAMP for 4h, and the amounts of IFNB1 or ISG15 transcripts were measured by RT-qPCR. c, Semi-native PAGE immunoblot analysis of STING dimerization and phosphorylation in DMSO and NHS-MTX (5 μM) pre-treated THP-1 monocytes stimulated with 100 μM 2’3’-cGAMP for 4h. For gel source data, see Supplementary Figure 1. d, DMSO and NHS-MTX (5 μM) treated THP-1 monocytes were treated with 100 μM 2’3’-cGAMP in the presence and absence of digitonin (5 μg/mL) for 4h, and the induction of IFNB1 mRNA was measured by RT-qPCR. e, f, DMSO (◯) and NHS-MTX (◯) (5 μM) pre-treated THP-1 monocytes (e) or K562 cells (f) were stimulated for 4h with 100 μM 2’3’-cGAMP, or not, in the presence or absence of digitonin (5 μg/mL), and the induction of OASL and ISG15 mRNA was measured by RT-qPCR. In panels b, d and e, data are means of n=2 technical replicates and data are representative of three independent experiments with similar results. In panel c, data are representative of three independent experiments with similar results.