Journal: Nature Communications

Article Title: Small molecule inhibition of cGAS reduces interferon expression in primary macrophages from autoimmune mice

doi: 10.1038/s41467-017-00833-9

Figure Lengend Snippet: Small-molecule inhibition of cGAS-dependent interferon induction in cellular assays. RU.365, RU.332, and RU.521 were tested in cellular assays for their effectiveness in inhibiting cGAS activity in dsDNA-stimulated RAW macrophages. The inhibition of type I IFN response via dsDNA was monitored via an interferon sensitive promoter coupled to a luciferase gene. The concentration response curves for RU.365 ( a ), RU.332 ( b ), and RU.521 ( c ) in RAW cells are shown. Error bars represent SEM

Article Snippet: Mouse RAW macrophages (RAW-Lucia ISG, RAW-Lucia ISG-KO-TREX1, RAW-Lucia ISG-KO-cGAS, and RAW-Blue Invivogen) were cultured in DMEM with high glucose, l -glutamine, phenol red, and sodium pyruvate (Life Technologies) supplemented with 10% FBS, 100 U ml –1 penicillin-streptomycin, Normocin (100 µg ml –1 , Invivogen), and Zeocin (200 µg ml –1 , Invitrogen) and an additional 20 mM l -glutamine and 1 mM sodium pyruvate.

Techniques: Inhibition, Activity Assay, Luciferase, Concentration Assay

Journal: Nature Communications

Article Title: Small molecule inhibition of cGAS reduces interferon expression in primary macrophages from autoimmune mice

doi: 10.1038/s41467-017-00833-9

Figure Lengend Snippet: Potent and selective inhibition of cGAS activity in RAW macrophage and BMDM cells from an Aicardi-Goutières Syndrome mouse model. RAW luciferase reporter cells were exposed to dsDNA ( a ), 5′ppp-HP20 RNA ( b ), Pam3CSK4 ( c ), poly(I:C) ( d ), lipopolysaccharide (LPS) ( e ), or recombinant murine interferon-β ( Ifnb ) f to promote either a type I interferon ( a ), ( b ), ( f ), or NF-κB ( c ), ( d ), ( e ) response under different immune stimuli and simultaneously treated with indicated small molecule (or vehicle). Type I interferon response was read via luciferase reporter, while NF-κB response was read via qRT-PCR. g RAW KO-cGAS reporter cells were exposed to dsDNA or cGAMP and simultaneously treated with each of the small molecules. h The cytotoxic effects of the lead compounds were tested in RAW macrophages at concentrations spanning the range tested in the dose response curves. Cytotoxicity was measured by the quantitation of ATP (CellTiter-Glo assay) at 72 h and shown as percent viability of cells. i BMDM cells from Trex1 −/− mice were treated with the indicated compound for 24 h and then harvested for qRT-PCR analysis. Shown are the results, relative to IFNB1 expression in Trex1 wild-type BMDMs. Error bars represent SEM

Article Snippet: Mouse RAW macrophages (RAW-Lucia ISG, RAW-Lucia ISG-KO-TREX1, RAW-Lucia ISG-KO-cGAS, and RAW-Blue Invivogen) were cultured in DMEM with high glucose, l -glutamine, phenol red, and sodium pyruvate (Life Technologies) supplemented with 10% FBS, 100 U ml –1 penicillin-streptomycin, Normocin (100 µg ml –1 , Invivogen), and Zeocin (200 µg ml –1 , Invitrogen) and an additional 20 mM l -glutamine and 1 mM sodium pyruvate.

Techniques: Inhibition, Activity Assay, Luciferase, Recombinant, Quantitative RT-PCR, Quantitation Assay, Glo Assay, Expressing