Journal: European journal of immunology
Article Title: Suppressive oligodeoxynucleotides containing TTAGGG motifs inhibit cGAS activation in human monocytes
Figure Lengend Snippet: A151 inhibits cGAS-STING signaling via both sequence and backbone specific binding to cGAS. (A) HEK cGAS low or HEK cGAS high cells were co-cultured with HEK STING cells for 24 h. Cultures were transfected with dsDNA (4 μg/ml) and/or treated with A151 (1 μM) where indicated. STING aggregate formation (mCherry clustering) in HEK STING cells was examined by fluorescence microscopy in three independent visual fields per well (containing > 100 cells/field) from four independent experiments. (B) To examine both sequence and backbone composition of A151-mediated cGAS inhibition, cells were pretreated with increasing amounts of A151, A151 (PD) or C151 and then stimulated with dsDNA or cGAMP (2 μg/ml). IFN-β mRNA levels were measured 6h later by qPCR using 18s RNA as an endogenous control. Results are shown as the percent inhibition of dsDNA stimulated cells from one representative of two independent experiments performed in triplicates (C) Lysates from immortalized THP-1 cells were subjected to pulldown analysis using dsDNA without (lane 1) or with (lane 2–7) 3 ’ -biotinylation. Increasing amounts of A151 (0.1, 0.3, 1, 3 and 10 μg) were included in lanes 3–7. 3 ’ -biotinylated A151 was run in lane 8 and whole lysate in lane 9. Immunoblots were probed for the presence of cGAS. Results show one representative experiment of two independent experiments. Statistical analysis: unpaired Student’s test, *** p
Article Snippet: A 3’ -biotin tag was added to A151 or dsDNA for pulldowns. dsDNA isolated from herring sperm was purchased from Sigma-Aldrich (St. Louis, MO, USA). cGAMP (cyclic [G(2’,5’)pA(3’,5’)p]) was purchased from Invivogen (San Diego, CA, USA). mtDNA was isolated from human hepatoma cells (HepG2) using the Mitochondrial Isolation Kit for mammalian cells (Thermo Fisher Scientific, Darmstadt, Germany) according to the manufacturer’s instruction.
Techniques: Sequencing, Binding Assay, Cell Culture, Transfection, Fluorescence, Microscopy, Inhibition, Real-time Polymerase Chain Reaction, Western Blot