Journal: Nature Communications
Article Title: STING-dependent sensing of self-DNA drives silica-induced lung inflammation
Figure Lengend Snippet: Self-dsDNA release is central to silica-induced lung inflammation and type I IFN response. a – m Silica microparticles (1 mg/mouse, i.t.) or saline were administered in WT mice and parameters were analyzed on day 7. a Concentration of extracellular dsDNA in the acellular fractions of bronchoalveolar lavage fluid (BALF). b – d Tmem173 , Mb21d1, Ifnα, and Ifnβ transcripts in the lungs and normalized to Gapdh expression. e Lung CXCL10 determined by ELISA and IFN-αβ proteins quantified by multiplex immunoassay. f Correlation between extracellular dsDNA concentrations and type I IFN gene expression. g Annexin V/PI flow cytometry analysis pre-gated on singlet cells. h Correlation between dead cells and extracellular dsDNA. i Increased nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in the BALF after silica exposure. j Immunoblots of caspase 3 (Casp3), cleaved caspase 3 (c-Casp3), gasdermin D (GSDMD), MLKL, and phosphorylated-MLKL (p-MLKL), normalized to β-actin. Immunoblot quantifications of ( k ) c-Casp3, ( l ) c-GSDMD, and ( m ) p-MLKL. n – u Silica microparticles administered with DNase I (200 µg/mouse, i.p.) as indicated ( n ). o Extracellular dsDNA in BALF acellular fraction. p Lung immunoblots of phospho-STING, STING, phospho-TBK1, TBK1, phospho-IRF3, IRF3, caspase 3 (Casp3), cleaved caspase 3 (c-Casp3), MLKL, and phosphorylated-MLKL (p-MLKL), with β-actin as a reference. q Quantification of STING dimer, relative to β-actin. r STING immunoblots under 5% reducing (left) or 1% 2-mercaptoethanol seminative conditions (2-ME; right). s Lung confocal images of DNA dye Draq5 (cyan) and STING (red). Bars, 20 µm. t Pulmonary IFN-αβ and CXCL10. u Neutrophils, macrophages, and protein extravasation in the BALF. * p
Article Snippet: Flow cytometry The left lung lobe was harvested and lung cells were isolated by enzymatic digestion using liberase (20 μg/mL; Roche) and DNase I (5 mg/mL; Sigma-Aldrich) in a final volume of 5 mL of RPMI at 37°C for 30 min under agitation.
Techniques: Mouse Assay, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Flow Cytometry, Cytometry, Western Blot