Journal: Nature Materials

Article Title: STING agonist delivery by tumour-penetrating PEG-lipid nanodiscs primes robust anticancer immunity

doi: 10.1038/s41563-022-01251-z

Figure Lengend Snippet: a , Chemical structures of the parent CDN STING agonist ( 1 ), CDN prodrug ( 2 ), diacyl lipid (3) and CDN-PEG-lipid ( 4 ). b , Schematic of LND containing CDN-PEG-lipid. c , Negative stain transmission electron micrograph of LND-CDNs and histogram of measured LND diameters. Scale bar, 200 nm. This experiment was performed once. d , Dynamic light scattering analysis of LND-CDN (red) and liposome-CDN (blue) particle size distributions. e , f , Representative flow cytometry histograms showing uptake of fluorescent LND-CDN (red) or liposome-CDN (blue) by RAW-ISG cells (STING reporter cell line) ( e ) or MC38 tumour cells ( f ) following 24 h incubation at 37 °C with 5 µM CDN in each formulation. g , Dose–response curves showing STING activation in RAW-ISG reporter cells as measured by bioluminescence reporter relative to the vehicle-treated control following 24 h stimulation at 37 °C. Data are presented as mean values ± s.e.m. with n = 4 biologically independent samples for each concentration tested.

Article Snippet: STING activation in mouse RAW-Lucia ISG (Invivogen) was performed according to the manufacturer's suggested protocol.

Techniques: Staining, Transmission Assay, Flow Cytometry, Incubation, Formulation, Activation Assay, Concentration Assay

Journal: Nature Materials

Article Title: STING agonist delivery by tumour-penetrating PEG-lipid nanodiscs primes robust anticancer immunity

doi: 10.1038/s41563-022-01251-z

Figure Lengend Snippet: a , Schematic of the chemistry of enzymatic cleavage of the dipeptide linker and subsequent self-immolative linker reaction. b , RAW macrophages were incubated for 18 hr with parent CDN or LND-CDN at varying concentrations. Cells were washed, then lysed and lysates were probed for quantity of parent CDN recovered by liquid chromatograph MS/MS analysis. c , Human THP-1-ISG reporter cells were incubated with indicated concentrations of LND-CDN for 3 hr, washed into fresh medium, then cultured for an additional 21 hr, followed by measurement of interferon-stimulated gene reporter activation by luciferase expression (n = 2 independent biological samples for each concentration and mean±s.e.m. is plotted; RLU, relative light units).

Article Snippet: STING activation in mouse RAW-Lucia ISG (Invivogen) was performed according to the manufacturer's suggested protocol.

Techniques: Incubation, Tandem Mass Spectroscopy, Cell Culture, Activation Assay, Luciferase, Expressing, Concentration Assay

Journal: Nature Materials

Article Title: STING agonist delivery by tumour-penetrating PEG-lipid nanodiscs primes robust anticancer immunity

doi: 10.1038/s41563-022-01251-z

Figure Lengend Snippet: a , PEGylated LND with and without PEG molecules represented. b , PEGylated liposome without and with cross-sectional view of liposome interior. c , DLS analysis of LND size distributions before and after diffusion through 50 nm pore diameter membranes. d , LND-CDN or CDN-PEG-lipids were incubated with 10% serum in a dialysis cassette with a 5 KDa MWCO membrane, and STING activation bioactivity remaining in the sample (as assessed by activation of RAW-ISG reporter cells) was measured over time (n = 3 biologically independent samples per timepoint, mean±s.e.m. is plottted).

Article Snippet: STING activation in mouse RAW-Lucia ISG (Invivogen) was performed according to the manufacturer's suggested protocol.

Techniques: Diffusion-based Assay, Incubation, Membrane, Activation 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: 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

Journal: Frontiers in Immunology

Article Title: STING Signaling Drives Production of Innate Cytokines, Generation of CD8 + T Cells and Enhanced Protection Against Trypanosoma cruzi Infection

doi: 10.3389/fimmu.2021.775346

Figure Lengend Snippet: STING deficiency negatively impacts activation of IRF-dependent pathways and cytokine expression in response to T. cruzi infection. (A) Experimental procedure. (B) IRF-dependent luciferase activity of STING-KO and RAW264.7 ISG macrophages infected or exposed to heat-killed T. cruzi . (C–E) Real-time PCR analysis of IFN-β, IL-6, and IL-12 mRNA expression in STING-KO and RAW264.7 ISG macrophages infected or exposed to heat-killed T. cruzi . HPRT1 was used as housekeeping gene. Control, T. cruzi uninfected/unexposed STING-KO and RAW264.7 ISG macrophages; NS, no statistical significance. (B–E) Two-way ANOVA and Tukey’s multiple comparison test. Data are shown as mean ± SD. Experimental figure was created with BioRender.com .

Article Snippet: RAW264.7-Lucia™ ISG and RAW264.7-Lucia™ ISG-STING-KO macrophages ( Invivo Gen, Toulouse, France) were plated in 24-well plates (Corning) at a density of 10 5 cells per well in 500 µl of DMEM2 24 h before infection, exposure to heat-killed trypomastigotes, or transfections.

Techniques: Activation Assay, Expressing, Infection, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Comparison

Journal: Frontiers in Immunology

Article Title: STING Signaling Drives Production of Innate Cytokines, Generation of CD8 + T Cells and Enhanced Protection Against Trypanosoma cruzi Infection

doi: 10.3389/fimmu.2021.775346

Figure Lengend Snippet: Trypanosoma cruzi DNA activates STING-dependent signaling. (A) Experimental procedure. (B) IRF-dependent luciferase activity of nontransfected (NT) and T. cruzi DNA-transfected STING-KO and RAW264.7 ISG macrophages. (C–E) Real-time PCR analysis of IFN-β, IL-6, and IL-12 mRNA expression in nontransfected (NT) and T. cruzi DNA-transfected STING-KO and RAW264.7 ISG macrophages. HPRT1 was used as housekeeping gene. NS, no statistical significance. (B–E) Two-way ANOVA and Tukey’s multiple comparison test. Data are shown as mean ± SD. Experimental figure was created with BioRender.com .

Article Snippet: RAW264.7-Lucia™ ISG and RAW264.7-Lucia™ ISG-STING-KO macrophages ( Invivo Gen, Toulouse, France) were plated in 24-well plates (Corning) at a density of 10 5 cells per well in 500 µl of DMEM2 24 h before infection, exposure to heat-killed trypomastigotes, or transfections.

Techniques: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Expressing, Comparison