Ht dna

Viral nucleic acids or HT-DNA (Sigma-Aldrich, St. Louis, MO, USA) were transfected into MDDCs or PMA-differentiated THP1 using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) or in PHH and HepG2-hNTCP-derived cell lines using X-tremeGENE HP reagent (Merck, Darmstadt, Germany) according to the manufacturer’s instructions.
For MDDC transfection experiments (Figure 1), Table 3 indicates the copy number/cell of viral DNAs (DNA copy/cell, determined by qPCR) or of viral RNAs (expressed as cDNA-equivalent copy/cell, determined by RT-qPCR). The indicated copy numbers/cell refers to the undiluted nucleic acids. SeV RNAs, MVA-gfp DNA and HBV DNA from viral particles were quantified from undigested nucleic acid preparations. HBV DNA replication intermediates were quantified from RNAse-digested samples. HBV RNA from viral particles or from total HepAD38 RNAs were quantified from DNase-digested samples to avoid any DNA contamination, and a control without RT was performed.

HEK-293T, BS-C-1, RK-13, and BHK-21 cells were grown in Dulbecco modified Eagle medium (Life Technologies) supplemented with 10% heat-inactivated fetal calf serum (FCS; Seralab) and 100 U/ml penicillin plus 100 μg/ml streptomycin (Pen/Strep; Life Technologies). THP-1-IFIT-1-GLuc cells were a gift from Veit Hornung (University of Munich, Munich, Germany). These cells had been modified to express GLuc under the control of the IFIT-1 promoter (26 (link)). THP-1-IFIT-1-GLuc cells were grown in RPMI 1640 (Life Technologies) supplemented with 15% FCS and Pen/Strep. PMA (Santa Cruz Biotechnology) was dissolved in dimethyl sulfoxide at 10 mg/ml. HT-DNA (Sigma) was dissolved in water at 2 mg/ml. 2′3′-cGAMP (Invivogen) was dissolved in water at 50 mg/ml. Sendai virus was a gift from Steve Goodbourn (St. George's University of London, London, United Kingdom). VACV strains MVA, vv811, COP, and WR, as well as CPXV strain Brighton Red, were obtained from Geoffrey L. Smith (University of Cambridge, Cambridge, United Kingdom). ECTV strain Moscow was from Antonio Alcami (Centro de Biología Molecular Severo Ochoa, Madrid, Spain). MVA was grown and titrated in CEF by conventional plaque assay. All other viruses were expanded in RK-13 or BS-C-1 cells and titrated in BS-C-1 cells. All viruses were purified through a 36% sucrose cushion before use.

Protein A/G magnetic beads (HY-K0202) and BLEB (HY-13813) were purchased from MedChemExpress (South Brunswick, USA). BeyoMag streptavidin magnetic beads (P2151), cell lysis buffer for Western and IP (P0013), 4’6-diamidino-2-phenyl-indole (DAPI, C1002), and Hoechst (C1011) were purchased from Beyotime. HT-DNA was purchased from Sigma-Aldrich (D1626, St. Louis, USA). Poly (dA:dT) was purchased from InvivoGen (tlrl-patn, Hong Kong, China). TRIzol reagent (15596018), Lipofectamine 3000 (L3000015), and Lipofectamine RNAiMAX (13778150) were purchased from Thermo Fisher Scientific (Waltham, USA). Phenylmethanesulfonyl fluoride (PMSF, CP8651) and cocktail protease inhibitors (SL1086) were purchased from Coolabor (Beijing, China). 2 × SYBR Green qPCR Master Mix (S2014S) was purchased from Singabio (Tianjin, China).

DNA plasmids pcDNA3.1(+):GFP or pcDNA3.1(+):IFI16 were used as templates for the in vitro synthesis of capped and polyadenylated mRNA using the mMESSAGE mMACHINE T7 Transcription Kit (ThermoScientific). IFI16 −/− HaCaT cells were seeded at 1 × 10⁵ cells/ml on coverslips 24 h before transfection with 1 μg ml⁻¹GFP mRNA or IFI16 mRNA for 6 h using using 1 μl ml⁻¹ of Lipofectamine 2000 (Life Technologies). Cells were then stimulated with 5 μg ml⁻¹ HT DNA (Sigma) for 1 h.

Anti-p-IRF3 (ab76493) and anti-IRF3 (ab68481) were from Abcam; EGCG (E4143), RSVL (R5010) and HT-DNA (D6898) were from Sigma-Aldrich; Poly(I:C) (tlrl-pic) was from InvivoGen; Anti-G3BP1 (13057-2-AP) was from Proteintech Group; Plasmid DNA, used as the DNA stimulator, was an empty vector plasmid (pCDX-Tet-On) and amplified with PureYield Plasmid Midiprep System (A2492, Progema); Genomic DNAs were purified using StarSpin Animal DNA Kit (D111-01, GenStar); Anti-human cGAS and anti-human GAPDH antibodies were gifts from Dr. Tao Li at National Center of Biomedical Analysis, Beijing, China.