Poly 1 c

HEK293T cells were maintained in DMEM (Gibco) supplemented with 10% fetal calf serum (FCS, Labtech) and 100 U/ml penicillin and 100 μg/ml streptomycin (Pen/Strep; Gibco). THP-1 cells were maintained in RPMI (Gibco) supplemented with 10% FCS and Pen/Strep. THP-1-IFIT-1 luciferase reporter cells express Gaussia luciferase under the control of the endogenous IFIT1 promoter have been described (Mankan et al., 2014 (link)). THP-1 CRISPR control, cGAS-/- and MAVS -/- knock out cells have been described (Mankan et al., 2014 (link)). Nup358 depleted HeLa cells have been described (Schaller et al., 2011 (link)). Lipopolysaccharide, poly I:C and TNFα were obtained from PeproTech. Sendai virus was obtained from Charles River Laboratories. Herring-testis DNA was obtained from Sigma. cGAMP was obtained from Invivogen. NF-ĸB Lucia THP-1 reporter cells were obtained from Invivogen. All cell lines were tested negative for mycoplasma.

To detect the polyreactivity of mAbs, the ELISA and immunofluorescence-based assays were performed as previously described^{20 (link)}. For ELISA-based detection of polyreactivity, plates were coated with poly I:C (15 mg/ml) (Life Technologies), LPS (15 mg/ml) (Sigma), or recombinant human insulin (10 mg/ml) (Sino Biological) in carbonate buffer. Plates were blocked with PBS with 0.05% Tween. Antibodies were serial diluted and incubated on the plate for 1 h at 37 °C followed by detection with goat anti-human IgG Fc at 1:8000. Absorbances were measured at OD450. An influenza antibody CR9114 and an anti-dsDNA antibody 3H9 was used as a positive control. For immunofluorescence-based detection of polyreactivity, HEp-2 cells were plated the day before experiment. HEp-2 cells were fixed with 4% PFA for 10 min followed by permeabilization with 0.3% Triton X-100 in PBS for 15 min. Biotinylated antibodies were incubated with cells for 1 h at the concentration of 50, 10 and 2 μg/ml, respectively and a FITC-linked streptavidin was then incubated with the cells for 1 h. The immunofluorescence was read and analyzed by Operetta (PerkinElmer).

Dendritic cells (DC) were generated from blood CD14⁺ cells of control donors and CLL patients as previously described [14 (link)]. Briefly, PBMC were incubated with anti-CD14 coated nanobeads (Miltenyi Biotec, Bergisch Gladbach, Germany). After washing, cells were passed through MidiMACS columns and CD14⁺ cells were separated according to the manufacturer’s instruction. Cells were resuspended in RPMI medium. The purity of CD14⁺ cells (>95%) was determined by flow cytometry. Isolated CD14⁺ cells were cultured with recombinant human (rh) GM-CSF (50 ng/ml) (Peprotech, London, UK) and rhIL-4 (20 ng/ml) (Peprotech) for 5 days. After 4 days of culture, the peptides (10 ug/ml) (see below) were added. For maturation, rhTNF-α (20 ng/ml) (Peprotech) and poly IC (50 ug/ml) (Invitrogen) were added at day 5. The cells were cultured for further 2 days. Harvested cells had the morphology of mature DC with a phenotype of CD3^–, CD14^–, CD19^–, CD80⁺, CD86⁺ and HLA-II⁺ determined by flow cytometry [14 (link)].

The polyclonal antibody against STING was generated by immunizing rabbit with recombinant human STING (221–379 aa). The goat polyclonal antibody against SCAP was from Santa Cruz Biotechnology. The rabbit polyclonal antibody against SCAP was a gift from Dr. Xiongzhong Ruan (Chongqing medical university). hemagglutinin (HA), Myc, Ub, SREBP1/2 and IRF3 antibody were purchased from Santa Cruz Biotechnology. TBK1 antibody was from abcam. Tom20 antibody was from BD Biosciences. Flag and β-actin antibodies were obtained from Sigma-Aldrich. Phospho-IRF3 and Phospho-TBK1 antibody was from Cell Signaling Technology.
Poly(dA:dT) and lipopolysaccharide (LPS) was obtained from Sigma-Aldrich. Poly(I:C) was purchased from Invitrogen. Wild type HSV-1 and HSV-1-GFP were kindly provided by Dr. Wentao Qiao (Nankai University) and Dr. Chunfu Zheng (Suzhou University), respectively. Listeria monocytogenes (10403 serotype) was a gift from Dr. Youcun Qian (Institute of Health Sciences). TBK1 kinase inhibitor BX795 was purchased from InvivoGen. ISD (Interferon stimulatory DNA) was prepared by annealing equimolar amounts of sense and antisense DNA oligonucleotides at 95°C for 10 min before cooling to room temperature. Oligonucleotides used are as follows:

The luciferase reporter assay were performed as previously described.^{47 (link)} Briefly, HEK293T, A549, HepG2, or sg25 cells were transfected with a control plasmid or protein expression plasmids together with the luciferase reporter plasmids using Viafect TM Transfection Reagent (Promega, Madison, WI, USA) or Lipofectamine TM 2000 (Invitrogen, San Diego, CA, USA). After 8 h, the cells were then transfected with poly(I:C) (Invitrogen), and the promoter activity was measured with Dual-Luciferase® Reporter Assay System (Promega) 16 h later. The relative firefly luciferase activities are normalized to the Renilla luciferase.