Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of STING-induced mitochondrial Drp1/N-GSDMD-mediated MtDNA release alleviates Sepsis-induced lung injury

doi: 10.1007/s00018-025-05774-x

Figure Lengend Snippet: GSDMD signal was correlatively activated with STING during ALI. A UMAP projections illustrating various cell types from samples obtained from COVID-19 patients. B Hierarchical clustering of gene expression data related to STING (TMEM173 for STING) and pyroptosis. This expression data were sourced from the GEO database ( GSE145926 ). C Correlation heatmaps depicting the expression of STING-related and pyroptosis-related genes. D - F Representative results of H&E staining after a time course of LPS challenge ( n = 6), with quantifications of the lung injury score and the ratio of lung wet weight to dry weight provided on the right. Images are displayed at 200× magnification, with a scale bar of 100 μm. G - H Relative quantification of mitochondrial DNA (D-loop and COXI) in the BALF of mice following a time course of LPS challenge ( n = 6). I Western blot images showing the STING/TBK1/IRF3 pathway in lung tissues after a time course of LPS challenge ( n = 6). J Western blot images for the NLRP3/Caspase1/GSDMD/IL-1β pathway in lung tissues after a time course of LPS challenge ( n = 6). K - N Relative mRNA levels of IL-1β, IL-6, TNF-α, and IFN-β in lung tissues following a time course of LPS challenge ( n = 6). O - S Representative results from co-immunostaining of F4/80, STING, and N-GSDMD in lung tissues after a time course of LPS challenge ( n = 6), with quantifications shown below

Article Snippet: Phosphorylated IRF3 (pIRF3) , Abmart , TA2436 , 1:1000 , WB.

Techniques: Gene Expression, Expressing, Staining, Quantitative Proteomics, Western Blot, Immunostaining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of STING-induced mitochondrial Drp1/N-GSDMD-mediated MtDNA release alleviates Sepsis-induced lung injury

doi: 10.1007/s00018-025-05774-x

Figure Lengend Snippet: N-GSDMD translocation to mitochondria was attenuated by disulfiram during LPS challenge. A Analysis of total and mitochondrial N-GSDMD in THP-1 cells stimulated with LPS at various time points, using COXIV as a control for mitochondrial membrane enrichment ( n = 5). B Measurement of total and mitochondrial N-GSDMD in THP-1 cells exposed to different concentrations of LPS, with COXIV serving as a control for mitochondrial membrane enrichment ( n = 5). C Representative images from immunofluorescent staining showing N-GSDMD and TOMM20 following LPS or LPS plus ATP stimulation. D Measurement of N-GSDMD in Mitochondria and Plasma Membrane in THP-1 cells following LPS or LPS plus ATP stimulation, with COXIV and ATP5B serving as a control for Mitochondria and Plasma membrane enrichment. E - F Flow cytometry histograms illustrating anti-N-GSDMD staining in mitochondria, with quantification of the percentage of N-GSDMD-positive mitochondria displayed below. G - H Evaluation of mitochondrial N-GSDMD, STING, and p-STING levels in THP-1 cells after LPS stimulation and pretreatment with varying concentrations of DSF, with COXIV as a control for mitochondrial membrane enrichment ( n = 5). Quantifications are provided on the right. I Relative quantification of cytosolic mtDNA levels (ND-1, D-loop, COXIII, and Cytochrome B) in THP-1 cells stimulated with 1 µg/mL LPS for 4 h, following a 2-hour pretreatment with different concentrations of DSF ( n = 4). J Representative images showing PicoGreen and MitoTracker staining in THP-1 cells after LPS stimulation, with a pretreatment of 5 µM DSF. Scale bar = 5 μm. K - L Western blot results depicting the STING/TBK1/IRF3 pathway and GSDMD in THP-1 cells following LPS stimulation and pretreatment with various concentrations of DSF ( n = 5). Quantifications are shown on the right. M Relative mRNA levels of IL-1β, IL-6, and IFN-β in response to LPS stimulation after pretreatment with different concentrations of DSF ( n = 5)

Article Snippet: Phosphorylated IRF3 (pIRF3) , Abmart , TA2436 , 1:1000 , WB.

Techniques: Translocation Assay, Control, Membrane, Staining, Clinical Proteomics, Flow Cytometry, Quantitative Proteomics, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of STING-induced mitochondrial Drp1/N-GSDMD-mediated MtDNA release alleviates Sepsis-induced lung injury

doi: 10.1007/s00018-025-05774-x

Figure Lengend Snippet: Inhibition of STING abrogated mitochondrial translocation of N-GSDMD. A - B Western blot analyses displaying the STING/TBK1/IRF3 pathway in THP-1 cells subjected to varying durations of LPS stimulation ( n = 5). Quantifications are shown on the right. C - D Western blot results illustrating the STING/TBK1/IRF3 pathway in both shCtrl and shSTING THP-1 cells following LPS stimulation ( n = 5). Quantifications are provided on the right. E Relative mRNA expression levels of IL-1β, IL-6, and IFN-β in shCtrl and shSTING THP-1 cells upon LPS stimulation ( n = 5). F Measurement of mitochondrial membrane potential using flow cytometry with JC-1, where JC-1 aggregation indicates normal mitochondrial membrane potential on the y-axis, and JC-1 monomers reflect low membrane potential on the x-axis. G Relative quantification of cytosolic mtDNA levels (ND-1, D-loop, COXIII, and Cytochrome B) in shCtrl and shSTING THP-1 cells following LPS stimulation ( n = 4). H - K Analysis of total and mitochondrial N-GSDMD, STING and p-TBK1 in both shCtrl and shSTING THP-1 cells after LPS stimulation, with COXIV serving as a control for mitochondrial membrane enrichment ( n = 5). Quantifications are presented below. L Representative images from immunofluorescent staining for N-GSDMD and TOMM20 in shCtrl and shSTING THP-1 cells following LPS stimulation. Scale bar is set at 5 μm. Quantifications are displayed below

Article Snippet: Phosphorylated IRF3 (pIRF3) , Abmart , TA2436 , 1:1000 , WB.

Techniques: Inhibition, Translocation Assay, Western Blot, Expressing, Membrane, Flow Cytometry, Quantitative Proteomics, Control, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of STING-induced mitochondrial Drp1/N-GSDMD-mediated MtDNA release alleviates Sepsis-induced lung injury

doi: 10.1007/s00018-025-05774-x

Figure Lengend Snippet: Pharmacological inhibition of STING alleviated ALI via reducing mitochondrial fission. A - E Representative findings from H&E staining and immunofluorescent staining for STING and ROS in lung tissues subjected to LPS challenge, with or without H-151 pretreatment. Scale bar is set at 100 μm. Quantifications of the lung injury score, the ratio of lung wet weight to dry weight and STING and ROS fluorescent intensity are provided on the right. F - G Relative quantifications of mtDNA (D-loop and COXI) in the BALF of mice after LPS challenge, with or without H-151 pretreatment ( n = 6). H - K Relative mRNA expression levels of IL-1β, IL-6, TNF-α, and IFN-β in lung tissues after LPS challenge, with or without H-151 pretreatment ( n = 6). L Western blot analyses depicting the STING/TBK1/IRF3 pathway in lung tissues following LPS challenge, with or without pretreatment using H-151 ( n = 6). M Western blot images showing levels of GSDMD, N-GSDMD, MFN1, MFN2, and Drp1 in lung tissues following LPS challenge, with or without H-151 pretreatment ( n = 6). N Representative images illustrating co-immunostaining for Drp1 and N-GSDMD in lung tissues after LPS challenge, with or without H-151 pretreatment ( n = 6). Scale bar is set at 100 μm. Representative images depicting COXIV in lung tissues following LPS challenge, with or without H-151 pretreatment. Scale bar is set at 5 μm

Article Snippet: Phosphorylated IRF3 (pIRF3) , Abmart , TA2436 , 1:1000 , WB.

Techniques: Inhibition, Staining, Expressing, Western Blot, Immunostaining