thp 1 ko sting thpd kostg  (InvivoGen)

Journal: medRxiv

Article Title: Autoantibodies From Connective Tissue Diseases Penetrate Cells and Exert Functional Properties

doi: 10.64898/2026.06.29.26356321

Figure Lengend Snippet: To investigate the capacity of ANAs to reach their nuclear targets, engage intranuclear antigens, and elicit functional consequences, we used antibodies from multiple sources: IgG from patients with systemic sclerosis (SSc-IgG) and healthy controls (HC-IgG). Total IgG were isolated from ATA-positive SSc patients (ATA + -IgG); from a subset of these individuals, ATAs were affinity-purified and the corresponding ATAs-depleted IgG fractions were retained. Fluorescently labeled ATAs (fluo-ATAs) and HC-IgG (fluo-HC-IgG) were also prepared. Experiments were conducted across a range of model systems, including cellular models (fibroblasts, endothelial cells, THP-1 monocytes, and SSc-derived fibroblasts), skin biopsies, and organ explant models (skin and lung). Using confocal microscopy, live-cell imaging, STED nanoscopy, functional assays (including the REEAD assay), and omics approaches, we demonstrate that ANAs enter living cells, accumulate in the nucleus, and engage their intracellular antigen. Nuclear ATAs inhibit TOP1 catalytic activity, induce DNA damage, and activate type I interferon production via the cGAS–STING pathway. We further identify neonatal Fc receptor (FcRn)-dependent intracellular trafficking as a key determinant of ANA nuclear access and show that pharmacological FcRn blockade impairs ATAs functionality.

Article Snippet: THP-1 WT, STING knockout (KO) (Cat. (thpd-kostg)) and cGAS KO (Cat. (thpd-kocgas) cells were obtained from InvivoGen.

Techniques: Functional Assay, Isolation, Affinity Purification, Labeling, Derivative Assay, Confocal, Microscopy, Live Cell Imaging, Activity Assay, Ex Vivo, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, RNA Sequencing, Confocal Microscopy, Super-Resolution Microscopy, Western Blot

Journal: medRxiv

Article Title: Autoantibodies From Connective Tissue Diseases Penetrate Cells and Exert Functional Properties

doi: 10.64898/2026.06.29.26356321

Figure Lengend Snippet: (A) Transcriptomic workflow. Endothelial cells pre-treated with IFN-α (4 hours) were exposed to ATAs or HC-IgG for 6 hours followed by bulk RNA sequencing. (B) PCA of mRNA profiles in ATAs- or HC-IgG-treated endothelial cells. n = 3 per condition. (C) Volcano plot of DEGs in ATAs versus HC-IgG (adjusted P < 0.05; |log₂ FC| > 1). (D) Heatmap of top 25 dysregulated mRNAs across ATAs-, HC-IgG-, and NS-treated cells (adjusted P < 0.05; |log₂ FC| > 1). (E) Pathway enrichment of mRNAs upregulated in ATAs versus HC-IgG; ORA (y-axis) and GSEA (x-axis, Reactome); five most significant labeled. (F) Western blot of p-STING in endothelial cells exposed to ATAs for 0-48 hours; cGAMP as positive control. (G) IFN-β (left) and CXCL10 (right) mRNA in endothelial cells exposed to HC-IgG or ATAs. n = 8 per condition. (H) Bioactive type I IFN in HEK-Blue IFN-α/β reporter cell supernatants treated with cGAMP, HC-IgG, or ATAs. n = 4 per condition. (I) CXCL10 protein in wild-type, cGAS-KO, and STING-KO THP-1 supernatants exposed to cGAMP, HC-IgG, or ATAs; attenuated in STING-KO cells. n = 4 per condition. (J) IFN-β mRNA in human lung explants exposed to HC-IgG, ATAs, or cGAMP for 6 or 24 hours. n = 4 per condition. NS: non-stimulated. Concentrations: 10 µg/mL HC-IgG and ATAs (all panels); cGAMP 50 µg/mL (F), 16 µg/mL (H-J). Box plots show median and full range. *P < 0.05, **P < 0.01; Kruskal-Wallis with Dunn’s post-hoc test (G, H, I, J).

Article Snippet: THP-1 WT, STING knockout (KO) (Cat. (thpd-kostg)) and cGAS KO (Cat. (thpd-kocgas) cells were obtained from InvivoGen.

Techniques: Bulk RNA Sequencing, Labeling, Western Blot, Positive Control, RNA Sequencing

Journal: medRxiv

Article Title: Autoantibodies From Connective Tissue Diseases Penetrate Cells and Exert Functional Properties

doi: 10.64898/2026.06.29.26356321

Figure Lengend Snippet: ANAs enter living cells, accumulate in the nucleus, and engage their intracellular targets. In the case of ATAs, nuclear accumulation leads to direct inhibition of topoisomerase I catalytic activity, induction of DNA damage, and activation of type I interferon production via the cGAS–STING pathway, ultimately promoting collagen deposition and fibrosis. The neonatal Fc receptor (FcRn) is identified as the key determinant of ANAs intracellular trafficking and nuclear access.

Article Snippet: THP-1 WT, STING knockout (KO) (Cat. (thpd-kostg)) and cGAS KO (Cat. (thpd-kocgas) cells were obtained from InvivoGen.

Techniques: Inhibition, Activity Assay, Activation Assay

Journal: Nature

Article Title: The mutational landscape of STING-induced immunity

doi: 10.1038/s41586-026-10685-3

Figure Lengend Snippet: a , Differentially expressed genes (DEGs) obtained from bulk RNA sequencing analysis of STING 1 knockout (SKO) THP-1 cells reconstituted with inducible EGFP-tagged STING WT, STING W119K/M120K, or STING D319K/D320K. Heatmap depicting the DEGs induced by D319K/D320K (2250 genes; FDR < 0.05, |log2 [fold change (FC)] | > 1; n = 3 per condition). To facilitate a direct comparison of the global transcriptional landscape, log2FC values for this identical gene set are plotted for the W119K/M120K mutant. Notably, the W119K/M120K mutant induces 39 DEGs (FDR < 0.05, |log2FC | >1; n = 3 per condition). b , Gene set enrichment analysis (GSEA) of pathways responsive to W119K/M120K and D319K/D320K expression, calculated using STRING. c , GSEA of pathways responsive to D319K/D320K expression, but not to W119K/M120K expression, calculated using STRING. d - g , mRNA expression levels of IFNB1 , IFIT1 and IFIT3 (ISGs), CCL3 and CCL4 (NF-κB-dependency) ( d , f ) or IL-8 production by ELISA ( e , g ) in SKO THP-1 cells reconstituted with inducible STING WT or STING W119K/M120K ( d , e ) or STING D319K/D320K ( f , g ) and treated with doxycycline for 24 h. Data are mean ± s.d. from three ( d , f ) and four ( e , g ) biological replicates. P values were calculated using Welch’s two-tailed t-test ( d - g ).

Article Snippet: THP-1-Dual STING 1 knockout cells were purchased from Invivogen (thpd-kostg).

Techniques: RNA Sequencing, Knock-Out, Comparison, Mutagenesis, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature

Article Title: The mutational landscape of STING-induced immunity

doi: 10.1038/s41586-026-10685-3

Figure Lengend Snippet: a , Violin plot depicting the average IFN scores of STING variants categorized in ClinVar database. B/BL, benign or benign like; P/PL, pathogenic or pathogenic like; VUS, variant of uncertain significance. b , Scatter plot depicting the average IFN and LC3 scores of STING variants characterized in the gnomAD database. c , IFN reporter activity of indicated Flag-tagged STING variants in IFN reporter cells seeded at 0.2 million per well before transfection. At least 10,000 cells were acquired per sample for flow cytometry analysis. d , IFN score of an index patient with heterozygous STING1 c.956A>G (p.Asp319Gly) variant. e , mRNA expression levels of IFNB1 , IFIT3 (ISGs) and CCL3 , CCL4 (NF-κB-dependent) in STING 1 knockout THP-1 cells reconstituted with inducible wild-type STING or D319G variant and seeded at 0.5 million per well, following DMSO or diABZI stimulation. f , Immunoblot analysis of pIRF3 and LC3B-I/II in IFN reporter cells transfected with plasmid encoding wild-type STING or D319G variant and treated with DMSO or diABZI. g , Scatter plot of STING mutations from the COSMIC database depicting mutation counts and IFN scores with cGAMP stimulation. The mutations with count of three or more and IFN score less than zero are highlighted in cyan. h , Kernel density estimation of COSMIC mutations (count three or more, cyan) and the entire mutational library (grey) corresponding to IFN scores with cGAMP. P values were calculated using the Mann–Whitney U -test. i , IFN reporter activity of indicated eGFP-tagged STING variants transfected into IFN reporter cells seeded at 0.2 million per well, followed by DMSO or diABZI treatment. Data are mean ± s.e.m. from three biological replicates ( c , e , i ). P values were obtained using one-way ANOVA followed by Tukey’s multiple-comparison test and ordinary two-way ANOVA ( c , e , i ).

Article Snippet: THP-1-Dual STING 1 knockout cells were purchased from Invivogen (thpd-kostg).

Techniques: Variant Assay, Activity Assay, Transfection, Flow Cytometry, Expressing, Knock-Out, Western Blot, Plasmid Preparation, Mutagenesis, MANN-WHITNEY, Comparison