Diabzi

Cells were treated with 2.5 μM diABZI (Selleckchem, S8796) and collected at the indicated time points. For cGAMP, 90mer and IVT4 stimulation, 0.1 μM cGAMP (Invivogen), 0.2 μg 90mer or 0.5 μg IVT4 was transfected using Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturer’s protocol, and cells were incubated for 3 h. The DNA sequences of 90mer is provided in Supplementary Table 1. Poly(I:C) (Invivogen) was added to the cell medium at a final concentration of 10 μg ml⁻¹ for 24 h. After infection with the HSV-1 KOS strain (MOI of 10), infected cells were incubated for 6 h. Pretreatment with BX795 (MedChemExpress) was performed at 2 µM for 24 h and pretreatment with bafilomycin A1 (Baf A1; Sigma) was performed at 20 nM for 1 h. MEFs treated with 5 μg ml⁻¹ or 40 μg ml⁻¹ DMXAA (Invivogen) were collected 2 h or 3 h after stimulation. For STING inhibition by H-151, H-151 (2 µM) was added into cells every 24 h for three days before cells were examined by RT–qPCR.

G150 (cat. no. HY‐128583; MedChemExpress, NJ, USA); H‐151 (cat. no. S6652, Selleck, Houston, TX, USA); TBK1/IKKε‐IN‐2 (cat. no. S0425, Selleck); Stattic (cat. no. S7024; Selleck); diABZI (cat. no. S8796; Selleck); G140 (cat. no. HY‐133916; MedChemExpress); RU.521 (cat. no. S6841; Selleck); TG003 (cat. no. S7320; Selleck), DOX (cat. no. S1208, Selleck); etoposide (cat. no. HY‐13629, MedChemExpress).

BJ, U2OS, and 293T cells were from ATCC and were authenticated through short tandem repeat (STR) profiling. All relevant authentication data are publicly available from ATCC. These cell lines differ in their growth rates and morphologies. All cell lines in this study were free of contaminations from other cell lines or mycoplasma. Cells were maintained with mycoplasma reagent and potential contaminations are regularly monitored through polymerase chain reaction (PCR) detection. All cells were cultured at 37 °C with 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 8% fetal bovine serum (FBS) and penicillin/streptomycin. Cyclic [G(2’,5’)pA(3’,5’)p] or cGAMP (#CT-CGMAP, ChemieTek) was dissolved in water and stored at −20 °C. The other two STING agonists diABZI (#S8796, Selleck) and C53 (#37354, Cayman) were dissolved in DMSO and stored in aliquots at −80 °C. Monensin (#16488, Cayman) was dissolved in ethanol and stored at −20 °C. cGAMP was delivered to cells using a mild digitonin buffer (50 mM HEPES pH 7.0, 100 mM KCl, 3 mM MgCl₂, 0.1 mM DTT, 85 mM sucrose, 0.2% BSA, 1 mM ATP, 8 μg/mL digitonin) (Woodward et al, 2010 (link)). Cells were treated with the buffer containing 1 μM cGAMP for 5-10 min and then changed back to the original culture media. All other chemicals were directly added to the cell culture media.