a, Overexpression of dinucleotide synthetases. HEK293T cells were transfected with different dinucleotide synthetases (100 ng) together with decreasing amounts of wild-type (WT) mmSTING or the R231A mutant (10, 5, 2.5, 1.25 and 0 ng) and a pIFNβ-luciferase reporter (pIFNβ-GLuc). Reporter activity was measured 16 h after transfection. RLU, relative light units. b, Direct stimulation with synthetic compounds. HEK293T cells were transfected with WT mmSTING or the R231A mutant in conjunction with pIFNβ-GLuc. The next day CMA was added or synthetic cyclic di-GMP or synthetic cGAMP(3′-5′) was transfected as indicated and pIFNβ-GLuc activity was assayed 16 h later. Representative data of two (a) or three (b) independent experiments are shown (mean values + s.e.m.).