Fig. 1.

BB-Cl-amidine inhibits STING-dependent signaling. (A) Structure of BB-Cl-amidine. (B and C) ELISA analysis of TNF-α and IFN-β in conditioned medium from BMDMs pretreated with vehicle control (DMSO) or BB-Cl-amidine (1 μM) for 1 h followed by treatment with the indicated ligands for 24 h. (D) ELISA analysis of IFN-β in conditioned medium from BMDMs pretreated with vehicle control (DMSO) or BB-Cl-amidine (1 μM) for 1 h followed by infection with HSV1 (MOI 10) or Sendai virus 20 (20 Units) for 24 h. (E) qPCR analysis of Ifnβ expression in BMDMs pretreated with the indicated concentrations of BB-Cl-amidine followed by treatment with diABZI-4 for 2 h. (F) ELISA analysis of IFNβ from BMDMs pretreated with the indicated concentrations of BB-Cl-amidine followed by treatment with diABZI-4 (500 nM) for 24 h. (G) Kinetic cell death analysis of primary BMDMs treated with the indicated concentrations of BB-Cl and stained with Sytox Orange and Hoechst. Cells were imaged using the Cytation5 microscope. One read per hour for 12 h. (H) Immunoblot analysis of phosphorylated STING, IRF3, TBK1, STAT1, P65, and LC3 conversion in whole-cell lysates from BMDMs pretreated with the indicated concentrations of BB-Cl-amidine for 1 h followed by treatment with diABZI-4 for 1 h. (I) qPCR analysis of Ifnβ in human primary monocytes pretreated with BB-Cl-amidine (1 μM) followed by treatment with 500 nM diABZI-4 for 2 h. (J and K) Serum analysis of IFN-β and TNF-α in mice administered BB-Cl-amidine (10 mg/kg) for 1 h followed by diABZI (0.5 mg/kg) for 5 h. B, C, and G representative data. D–F, and H pooled data from three independent experiments. I and J vehicle (n = 4), diABZI-4 (n = 6), diABZI-4 + BB-Cl-amidine (n = 5). *P < 0.05; **P < 0.001. Error bars show mean ± SEM.