Tlrl bx7

2′3′-cGAMP was purchased from Invivogen. 2′3′-cGAMP (5 µg/ml) (Invivogen, tlrl-nacga23-02) was added to the cells complexed with LyoVec (Invivogen, Lyec-12) in order to aid internalization. ISD (Interferon stimulatory DNA)/LyoVec (Invitrogen, tlrl-isdc, 1 µg) was also used. Membrane lipid strips were obtained from Echelon. The following inhibitors were used: Fura-2 AM, Ca2+ selective fluorescent indicator (Abcam, ab120873), BX795 (TBK-1 inhibitor, Invivogen, tlrl-bx7, 1 µM), RU.521 (cGAS inhibitor, Invivogen, inh-ru521, 500 nM). GSK2998533 (100 nM) & GSK2683449 (100 nM) were provided by GlaxoSmithKline (GSK). PIK93 was also used (Sigma Aldrich, 1 µM)

Bat and human organoids were sheared using a glass Pasteur pipette and incubated in a basal medium (Advanced DMEM/F-12 (Gibco) supplemented with 1% HEPES, 1% GlutaMAX and 1% Penicillin/Streptomycin) with 10 µg/ml Poly(I:C) (InvivoGen, tlrl-pic-5) or DMSO in a suspension plate. At the indicated time points post-treatment, treated or mock-treated organoids were harvested and subjected to RT-qPCR assay and Western blot. The cell-free media were collected and applied to ELISA and PRM-MS. For pathway inhibition experiments, bat and human intestinal organoids in the basal medium were pretreated with 1 µg/ml or 0.1 µg/ml CYT 387 (InvivoGen, inh-cy87), or 1 µM or 0.1 µM BX795 (InvivoGen, tlrl-bx7), or mock-treated with 0.1% DMSO overnight. Subsequently, the organoids were sheared mechanically and incubated with or without 10 µg/ml Poly(I:C) and the inhibitors with the initial concentrations in a suspension plate. At the indicated time points, the organoids were collected to detect the expression levels of antiviral genes by RT-qPCR assay. Cell-free media were harvested and applied to ELISA and PRM-MS.

Following drugs were used as indicated in the corresponding sections: H151 (STING-inhibitor, 1 µM, 5 h for WB/IF, Invivogen), tlrl-bx7 (TBK1/IKKε inhibitor, 1 µM, 2-4-6-24 h for WB, 5 h for IF, Invivogen), cGAMP (STING-ligand/activator, 2 µg/mL, 2-4-6-24-48 h for WB, 5 h for IF, Invivogen), TLR-BW006 Class B CpG oligonucleotide (TLR9-ligand/activator, 2.5 µM, 2-4-6-24-48 h for WB, 5 h for IF, Invivogen), ODN-INH-18 Class B inhibitory (TLR9-inhibitor, 2.5 µM, 2-4-6-24-48 h for WB, 5 h for IF, Invivogen), and bafilomycin (vascular H⁺-ATPase inhibitor, 200 nM, 6 h for IF, BioConnect).