Mice
By further mating the male and female Trex1+/− mice(Purchased from jackson lab), Trex1−/− mice were generated and genotyped by standard PCR. Six-to-eight-week-old wild-type (WT) were purchased from specific pathogen-free Biotechnology Company Limited (Beijing, China). Mice were on the C57BL/6 background throughout this study. Experiments on animals were conducted in strict compliance with regulations specified in the Guide for the Care and Use of Laboratory Animals issued by The Chinese People’s Liberation Army General Hospital's Fifth Medical Centre.
Cell culture
A femoral bone marrow sample of eight-week-old female C57BL/6 mice was collected for the isolation of bone marrow-derived macrophages (BMDMs). For the culture of BMDMs, 10% fetal bovine serum, 1% penicillin, and streptomycin (P/S) were added to Dulbecco's Modified Eagle's Medium (DMEM). The cultures of human leukemic monocytes (THP-1 cells) were performed in RPMI 1640 medium
Reagents and antibody
polyinosinic–polycytidylic acid (poly(I:C)), Herring testis (HT) DNA were purchased from Sigma-Aldrich (Jefferson City, USA). 2'3'-cGAMP was purchased from InvivoGen. StarFect High-efficiency Transfection Reagents were purchased from GenStar. Licochalcone B (HY-N0373), DMXAA(HY-10964), Cridanimod (HY-W011890 ) and diABZI STING agonist-1 trihydrochloride (HY-112921B) were purchased from Med Chem Express (State of New Jersey, US). Rabbit monoclonal anti-Phospho-IRF-3 (86691) was purchased from Gene Tex (China). Rabbit monoclonal anti-Phospho-IRF-3 (76439) was purchased from Abcam. Anti-Flag M2 (F3165), TMEM173/STING Polyclonal antibody (19851-1-AP) were purchased from Cell Signaling Technology (China).StarScript III All-in-one RT Mix with gDNA Remover (A23010) are from GenStar.
Cell counting Kit 8 assay
Cell viability assays were performed utilizing the Cell Counting Kit 8 (CCK-8). BMDMs were incubated at a density of 106 cells/ml overnight at 37°C in 96-well plates. The cells were treated with varying concentrations of Licochalcone B for 24 hours, and optical density was measured at a wavelength of 450 nm after each incubation.
Real-time PCR
After the isolation of total RNA with TRIzol reagent (Invitrogen), reverse transcription was performed. SYBR GREEN MASTER MIX (GenStar ,A304-10) was used to label the amplified gene transcripts. Supplementary Table 1 presents a list of primers used for amplifying the target genes.
Enzyme-linked immunosorbent assay (ELISA)
The levels of IFN-β in the supernatants from cell cultures were quantified using the IFN-β Bioluminescent ELISA kit (luex-mifnbv2; InvivoGen), according to the manufacturer's instructions.
Western blotting
Western blot was performed on the protein from the cell culture lysate, as described previously [24]. For determination of the expression of p-IRF3, STING, and IRF3 in cell lysates, immunoblots were conducted with HSP90 serving as a loading control. A wet-transfer system was used to transport the protein samples after they were boiled for 15 min at 105°C. In the second step, at a temperature of 4°C, the membranes were incubated with primary antibodies overnight after being pre-incubated with 5% fat-free milk for 1 hour. As a secondary antibody, the horseradish peroxidase-labeled antibody was applied to the blots. Finally, a darkroom was used for developing and fixing X-ray films after they were made photosensitive.
Immunofluorescence
THP-1 cells were fixed with 4% paraformaldehyde, permeabilized in 0.25% Triton X-100, and subsequently blocked with 5% Rapid blocking solution (Beijing, China, C200501) for 1 hour. Primary antibodies were added, and the cells were then stained and incubated with fluorescent-conjugated secondary antibodies. DAPI was employed to counterstain the nuclei.
STING oligomerization assay
Assays for STING oligomerization were conducted as described previously[25]. In brief, native sample buffer was used to load cell lysates onto a native-PAGE gel, which was pre-run in an electrophoresis buffer (cathode and anode chambers were respectively filled with 25 mM Tris-Cl, pH 8.4, 192 mM glycine with or without 5% deoxycholate) for 30 minutes at 100 mA and then electrophoresed for 50 minutes at 25 mA. Similarly, SDS electrophoresis buffer (25 mM Tris pH 8.3, 250 mM glycine, 0.1% SDS) was added to the gel, and the gel was allowed to soak for 30 minutes at room temperature, followed by immunoblotting with an anti-STING antibody.
Immunoprecipitation and pull-down
Immunoprecipitation and pull-down analysis were performed on proteins extracted from cell culture lysates [19]. Cultured HEK-293T cells were seeded into six-well cell culture plates and allowed to grow overnight. Subsequently, cells were first transfected with a plasmid (Flag\HA-vector, Flag-IRF3, HA-STING, Flag-TBK1) for 24 h, and then treated with LicoB for 6 h. Using the RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% Triton X-100, 1% sodium deoxycholate) and complete protease inhibitors (Protease inhibitors Cocktail, TargetMol, C0001) cells were lysed. After incubation with anti-Flag-M2 beads, the supernatant was t rotated and washed with the RIPA cracking buffer five times. The pulled target protein was then analyzed with a western blot assay. For the pull-down experiment, LicoB was coupled with CNBr-activated Sepharose 4B(Sigma-Aldrich 68987-32-6) overnight at 37°C. THP-1 (5 ×106 cells /ml) of the PMA primer was inoculated in Petri dishes overnight and incubated for 2 h with or without HT-DNA. The supernatant was discarded, and the cells were lysed in the RIPA lysis buffer containing complete protease inhibitors. Sepharose 4B conjugated with LicoB was incubated overnight at 4℃, spun, and washed with the RIPA buffer. The pulled target was analyzed with a western blot assay.
Mice and in vivo studies
Female WT mice (8–10 weeks of age) were injected with LicoB (40 mg/kg) or vehicle (5% DMSO + 5% Tween 80 + normal saline) for assessment of its in vivo inhibitory effect. The concentration of 180 mg/kg CMA was administered after 1 h. Four h later, the peritoneal lavage fluid (the peritoneal cavity was washed with 10 ml ice-cold 1× PBS) and serum were collected from euthanized mice, to measure cytokine levels (IFN-β, IL-6, and TNF-α) induced by CMA. Female WT or Trex1−/− mice (four-week-old) were injected intraperitoneally with LicoB (40 mg/kg) or vehicle (5% DMSO + 5% Tween80 + normal saline) for 15 consecutive days. Following the mice were euthanized, and heart, liver tissues, lungs, tongue, intestine, stomach, kidney, and muscle were collected for mRNA analysis. Hematoxylin–eosin (H&E) staining techniques were used to determine the histopathology in mice tissues.
Statistical analyses
To compare between the two groups, an unpaired t-test was utilized. For comparison among multiple groups, either a one-way ANOVA with Dunnett’s post-hoc test or Kruskal–Wallis test was applied, using GraphPad Prism 6.0 software. The data are presented as the mean ± standard error of the mean (SEM). A P-value of less than 0.05 was considered statistically significant.