2 3 cgamp

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)

Cells were seeded at 1–1.5 × 10⁵ cells per ml 24 h before transfection, and stimulated with 1 μg ml⁻¹ HT DNA (HT DNA, Sigma), a double-stranded 70mer oligonucleotide derived from VACV (5′-CCATCAGAAAGAGGTTTAATATTTTTGTGAGACCATCGAAGAGAGAAAGAGATAAAACTTTTTTACGACT-3′)10 (link), Y-G3 DNA (5′-GGGGAACTCCAGCAGGACCATTGGGG-3′) or Y-C3 DNA (5′-CCCGAACTCCAGCAGGACCATTGCCC-3′)24 (link). DNA oligonucleotides were synthesized by Biofins Genomics, Germany. In vitro transcribed RNA containing a 5′-triphosphate was generated using the MEGAScript T7 transcription kit (Thermo Fisher) with pcDNA3.1: EGFP as template. 50 ng ml⁻¹ of in vitro transcribed RNA and 100 ng ml⁻¹ poly(I:C) (Sigma) were used, unless indicated otherwise. 2′3′ cGAMP (Invivogen) or cyclic di-AMP (Invivogen) were transfected at 20 and 100 μg ml⁻¹, respectively. All transfections were carried out with 1 μl Lipofectamine 2000 (Life Technologies) per ml medium.
Transfection by digitonin permabilization was carried out in a buffer containing 50 mM HEPES (pH 7), 100 mM KCl, 3 mM MgCl₂, 0.1 mM DTT, 85 mM saccharose, 1 mM ATP, 0.1 mM GTP and 0.2% (v/v) BSA. 25 μg ml⁻¹ HT DNA (Sigma). 15 μM 2′3′ cGAMP or 2′3′ cGAM(PS)₂ (both Invivogen) was transfected using 5 μg ml⁻¹ digitonin in permeabilization buffer for 10 min at 37 °C before replacing the permabilization buffer with DMEM containing 10% (v/v) FCS.