Journal: Molecular Medicine

Article Title: Loss of XRCC1 promotes cGAS/STING mediated innate immune signaling in gastric cancer

doi: 10.1186/s10020-026-01429-0

Figure Lengend Snippet: ATM inhibitors enhance cGAS/STING signal activation. A STING/TBK1/IRF-3 signaling pathway activation detected by Western blot of protein extract from siRNA control (WT) and siRNA-XRCC1 knockdown cells treated with and without ATM inhibitor. Anti-IRF3/ p-IRF3 (ser396); TBK1/P-TBK-1 (Ser172) antibodies were obtained from Cell Signaling. Note that fraction of phosphrylated value calculated obtained by dividing the band intensity of physphrylated proteins relative to the total respective unphosphorylated protein band intensity. All band intensity were measured using Bio-Rad Image lab software. B Enrichment of cytosolic DNA sensing genes in WT and XRCC1 knockout GC cells from HallMark data base in enrichment score Gene Set Enrichment Analysis (GSEA ver. 4.3.2) in GSEA; C Enrichment of cytosolic DNA sensing genes expression in GC cells were treated with 10 µM ATM inhibitor (ATMi, KU55933) for 24 h. D Volcano graph represent the overall gene expression of inflammatory, cytosolic DNA sensing, and inflammatory cell signaling ; E Heat map of set of genes in volved in interferon response in XRCC1 proficient NCI-N87 (N87-WT) and deficient NCI-N87 (N87-KD)

Article Snippet: Next, the membranes were blocked with 5% BSA for 1 h, and then incubated with primary antibodies against STING (Cat. 13647 S, Cell Signaling), IRF3 (Cat. 4302 S, Cell Signaling), p-IRF3 (Cat. 4947 S, Cell Signaling), TBK1 (Cat. 3013 S, Cell Signaling), p-TBK1 (Cat. 5483 S, Cell Signaling), PD-L1 (Cat. 13684, cell signaling), β-actin (Cat. 3700 S, Cell Signaling), and Vinculin (Cat. 13901 S, Cell Signaling) overnight at 4 °C.

Techniques: Activation Assay, Western Blot, Control, Knockdown, Software, Knock-Out, Expressing, Gene Expression

Journal: Molecular Medicine

Article Title: Loss of XRCC1 promotes cGAS/STING mediated innate immune signaling in gastric cancer

doi: 10.1186/s10020-026-01429-0

Figure Lengend Snippet: A - E Inflammation-mediated response in XRCC1 deficient mice stomach. A Experimental design of tamoxifen treatment to Cre expression and excised XRCC1 gene segment. B Representative image of gastric histopathology of XRCC1 deficient mice stomach with H and E staining; H & E stain of stomach sections from Cre-XRCC1 flox/flox (WT) and Cre+XRCC1 △/△ . C Quantification of inflammatory score between Cre-XRCC1 flox/flox (5 mice) and Cre+XRCC1 Δ/Δ mice (5 mice) stomach tissue sections; Low-to-medium-grade inflammation with abundant lymphocytes and neutrophilic granulocytes in XRCC1 deficient mice stomach. Note that inflammation score was done by grading pathological severity as normal (0), mild (Kelley and Duggan ), moderate (Sung et al. ), severe (Dong et al. ); D Western blot analysis of TBK1,p-TBK1(Ser172), IRF3 and p-IRF3 in stomach tissue from Cre-XRCC1 flox/flox versus Cre+XRCC1 Δ/Δ mice. Note that fraction of phosphrylated value calculated obtained by dividing the band intensity of physphrylated proteins relative to the total respective unphosphorylated protein band intensity (ratio). E mRNA expression of genes of cytokines and chemokines quantified using RT-qPCR from Cre-XRCC1 flox/flox versus Cre+XRCC1 Δ/Δ

Article Snippet: Next, the membranes were blocked with 5% BSA for 1 h, and then incubated with primary antibodies against STING (Cat. 13647 S, Cell Signaling), IRF3 (Cat. 4302 S, Cell Signaling), p-IRF3 (Cat. 4947 S, Cell Signaling), TBK1 (Cat. 3013 S, Cell Signaling), p-TBK1 (Cat. 5483 S, Cell Signaling), PD-L1 (Cat. 13684, cell signaling), β-actin (Cat. 3700 S, Cell Signaling), and Vinculin (Cat. 13901 S, Cell Signaling) overnight at 4 °C.

Techniques: Expressing, Histopathology, Staining, Western Blot, Quantitative RT-PCR

Journal: Molecular Medicine

Article Title: Loss of XRCC1 promotes cGAS/STING mediated innate immune signaling in gastric cancer

doi: 10.1186/s10020-026-01429-0

Figure Lengend Snippet: ATM inhibitors enhance cGAS/STING signal activation. A STING/TBK1/IRF-3 signaling pathway activation detected by Western blot of protein extract from siRNA control (WT) and siRNA-XRCC1 knockdown cells treated with and without ATM inhibitor. Anti-IRF3/ p-IRF3 (ser396); TBK1/P-TBK-1 (Ser172) antibodies were obtained from Cell Signaling. Note that fraction of phosphrylated value calculated obtained by dividing the band intensity of physphrylated proteins relative to the total respective unphosphorylated protein band intensity. All band intensity were measured using Bio-Rad Image lab software. B Enrichment of cytosolic DNA sensing genes in WT and XRCC1 knockout GC cells from HallMark data base in enrichment score Gene Set Enrichment Analysis (GSEA ver. 4.3.2) in GSEA; C Enrichment of cytosolic DNA sensing genes expression in GC cells were treated with 10 µM ATM inhibitor (ATMi, KU55933) for 24 h. D Volcano graph represent the overall gene expression of inflammatory, cytosolic DNA sensing, and inflammatory cell signaling ; E Heat map of set of genes in volved in interferon response in XRCC1 proficient NCI-N87 (N87-WT) and deficient NCI-N87 (N87-KD)

Article Snippet: Next, the membranes were blocked with 5% BSA for 1 h, and then incubated with primary antibodies against STING (Cat. 13647 S, Cell Signaling), IRF3 (Cat. 4302 S, Cell Signaling), p-IRF3 (Cat. 4947 S, Cell Signaling), TBK1 (Cat. 3013 S, Cell Signaling), p-TBK1 (Cat. 5483 S, Cell Signaling), PD-L1 (Cat. 13684, cell signaling), β-actin (Cat. 3700 S, Cell Signaling), and Vinculin (Cat. 13901 S, Cell Signaling) overnight at 4 °C.

Techniques: Activation Assay, Western Blot, Control, Knockdown, Software, Knock-Out, Expressing, Gene Expression

Journal: Molecular Medicine

Article Title: Loss of XRCC1 promotes cGAS/STING mediated innate immune signaling in gastric cancer

doi: 10.1186/s10020-026-01429-0

Figure Lengend Snippet: A - E Inflammation-mediated response in XRCC1 deficient mice stomach. A Experimental design of tamoxifen treatment to Cre expression and excised XRCC1 gene segment. B Representative image of gastric histopathology of XRCC1 deficient mice stomach with H and E staining; H & E stain of stomach sections from Cre-XRCC1 flox/flox (WT) and Cre+XRCC1 △/△ . C Quantification of inflammatory score between Cre-XRCC1 flox/flox (5 mice) and Cre+XRCC1 Δ/Δ mice (5 mice) stomach tissue sections; Low-to-medium-grade inflammation with abundant lymphocytes and neutrophilic granulocytes in XRCC1 deficient mice stomach. Note that inflammation score was done by grading pathological severity as normal (0), mild (Kelley and Duggan ), moderate (Sung et al. ), severe (Dong et al. ); D Western blot analysis of TBK1,p-TBK1(Ser172), IRF3 and p-IRF3 in stomach tissue from Cre-XRCC1 flox/flox versus Cre+XRCC1 Δ/Δ mice. Note that fraction of phosphrylated value calculated obtained by dividing the band intensity of physphrylated proteins relative to the total respective unphosphorylated protein band intensity (ratio). E mRNA expression of genes of cytokines and chemokines quantified using RT-qPCR from Cre-XRCC1 flox/flox versus Cre+XRCC1 Δ/Δ

Article Snippet: Next, the membranes were blocked with 5% BSA for 1 h, and then incubated with primary antibodies against STING (Cat. 13647 S, Cell Signaling), IRF3 (Cat. 4302 S, Cell Signaling), p-IRF3 (Cat. 4947 S, Cell Signaling), TBK1 (Cat. 3013 S, Cell Signaling), p-TBK1 (Cat. 5483 S, Cell Signaling), PD-L1 (Cat. 13684, cell signaling), β-actin (Cat. 3700 S, Cell Signaling), and Vinculin (Cat. 13901 S, Cell Signaling) overnight at 4 °C.

Techniques: Expressing, Histopathology, Staining, Western Blot, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: TAX1BP1 and A20 Inhibit Antiviral Signaling by Targeting TBK1-IKKi Kinases

doi: 10.1074/jbc.m110.109819

Figure Lengend Snippet: FIGURE 4. TAX1BP1 and A20 target TBK1-IKKi for deubiquitination. A, 293T cells were cotransfected with epitope-tagged constructs as indicated (1 g of FLAG-TBK1, FLAG-A20 WT, FLAG-A20 C103A, and FLAG A20 1–367 and 500 ng of HA-Ub Lys63-only). Co-IPs and immunoblots evaluating TBK1 ubiquitination and immu- noprecipitation were performed using the indicated antibodies. B, epitope-tagged constructs (1 g of FLAG- IKKi,500ngofHA-Ub,and1or2gofGFP-TAX1BP1)weretransfectedinto293Tcells.Co-IPsandimmunoblots evaluating IKKi ubiquitination were performed as in A. C, 293T cells were transfected with control scrambled siRNA or siRNA targeting TAX1BP1. After 24 h, cells were cotransfected with the indicated plasmids (1 g of FLAG-IKKi and 500 ng of HA-Ub Lys63-only), and IKKi polyubiquitination was determined as in B. D, Tax1bp1/ or Tax1bp1/ MEFs were mock-infected or virally infected for 16 h, and lysates were subjected to immuno- precipitation and/or immunoblotting as indicated. E, 293T cells were cotransfected with IFN luc (100 ng) and pRL-tk (10 ng) DNA and plasmids encoding TBK1 or IRF7 (1 g) and Lys63-only ubiquitin (500 ng) as indicated. WT, wild type; IB, immunoblot; IP, immunoprecipitation.

Article Snippet: Other antibodies used in this study were IRF3, GFP, TRAF6, and TRAF3 (Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)); phospho-IRF3 and Myc (Upstate); TBK1, TAX1BP1, and -actin (Abcam); A20 (BD Biosciences); IKKi (Imgenex); ubiquitin (Stressgen); Lys63linked ubiquitin (Biomol); and HA (Roche Applied Science).

Techniques: Construct, Western Blot, Ubiquitin Proteomics, Transfection, Control, Infection, Immunoprecipitation