Journal: Cancers

Article Title: Nectin-1 Expression Correlates with the Susceptibility of Malignant Melanoma to Oncolytic Herpes Simplex Virus In Vitro and In Vivo

doi: 10.3390/cancers13123058

Figure Lengend Snippet: Correlation of the oncolytic activity of T-VEC with the expression of stimulator of interferon genes (STING) and cyclic GMP-AMP synthase (cGAS) in a panel of melanoma cell lines. ( A ) Western Blot analysis of STING and cGAS with respect to housekeeping protein ß-actin using lysates of 20 melanoma cell lines. ( B ) Densitometric quantification of STING and cGAS, normalized for ß-actin and shown as the mean and standard error of three independent Western blot experiments. ( C ) Spearman correlation coefficient, ( D ) box plot, and ( E ) ROC curve analyses of all responder and non-responder cell lines with respect to STING (upper part) and cGAS expression (lower part) and corresponding susceptibility to T-VEC induced cell death. p values for box plots were calculated using the Mann–Whitney test; ROC curves analyzed the area under the curve (AUC).

Article Snippet: The following primary antibodies were used: Rabbit monoclonal antibody against STING (D2P2F, Cell Signaling; dilution for patient samples 1:50, dilution for cell lines 1:200), murine monoclonal antibody against Nectin-1 (R1.302.12, Santa Cruz, Heidelberg, Germany; dilution for patient samples 1:50), rabbit polyclonal antibody against Nectin-1 (AB_2736197, Invitrogen/Thermo Fisher; dilution for cell lines 1:100), murine monoclonal antibody against HVEM (CW10, Santa Cruz; dilution 1:500 for patient samples and cell lines), rabbit polyclonal antibody against cGAS (NBP1-86761, Novus Biologicals/Bio-Techne, Wiesbaden Nordenstadt, Germany; dilution for patient samples 1:200), and rabbit monoclonal antibody against cGAS (D1D3G) (Cell Signaling; dilution for cell lines 1:200).

Techniques: Activity Assay, Expressing, Western Blot, MANN-WHITNEY

Journal: Cancers

Article Title: Nectin-1 Expression Correlates with the Susceptibility of Malignant Melanoma to Oncolytic Herpes Simplex Virus In Vitro and In Vivo

doi: 10.3390/cancers13123058

Figure Lengend Snippet: Correlation of biomarkers evaluated by flow cytometry, Western blot, and immunohistochemistry with the oncolytic activity of T-VEC in melanoma cell lines. Spearman correlation coefficient analysis for ( A ) Nectin-1 and HVEM expression, measured by flow cytometry (FACS) and immunohistochemistry (IHC), ( B ) STING and cGAS expression, evaluated by Western blot (WB) and immunohistochemistry, and ( C ) expression of all four biomarkers in immunohistochemistry with the oncolytic activity of T-VEC in 20 melanoma cell lines. Data show the mean of three independent experiments for each biomarker and MTT assay.

Article Snippet: The following primary antibodies were used: Rabbit monoclonal antibody against STING (D2P2F, Cell Signaling; dilution for patient samples 1:50, dilution for cell lines 1:200), murine monoclonal antibody against Nectin-1 (R1.302.12, Santa Cruz, Heidelberg, Germany; dilution for patient samples 1:50), rabbit polyclonal antibody against Nectin-1 (AB_2736197, Invitrogen/Thermo Fisher; dilution for cell lines 1:100), murine monoclonal antibody against HVEM (CW10, Santa Cruz; dilution 1:500 for patient samples and cell lines), rabbit polyclonal antibody against cGAS (NBP1-86761, Novus Biologicals/Bio-Techne, Wiesbaden Nordenstadt, Germany; dilution for patient samples 1:200), and rabbit monoclonal antibody against cGAS (D1D3G) (Cell Signaling; dilution for cell lines 1:200).

Techniques: Flow Cytometry, Western Blot, Immunohistochemistry, Activity Assay, Expressing, Biomarker Discovery, MTT Assay

Journal: Cancers

Article Title: Nectin-1 Expression Correlates with the Susceptibility of Malignant Melanoma to Oncolytic Herpes Simplex Virus In Vitro and In Vivo

doi: 10.3390/cancers13123058

Figure Lengend Snippet: Oncolytic effect of T-VEC upon injection into 35 malignant melanoma lesions. ( A ) Waterfall plot showing the response rate of each individual lesion as increase or decrease of the tumor volume, calculated as (length × width × thickness)/2 when the maximum anti-tumor effect had been achieved. ( B ) Based on this criterion, 14 and 21 lesions were respectively categorized as non-responders (+129.89% to −29%) and responders (−30% to −100%). ( C ) Representative example of Nectin-1, HVEM, STING, and cGAS immunostaining in a melanoma lesion responding (upper panel) or not responding (lower panel) to intratumoral T-VEC injection. Images provide an overview and details at higher magnification (inserts); corresponding size bars are included. ( D ) Spearman correlation coefficient analysis of Nectin-1, HVEM, STING, and cGAS immunostaining with the oncolytic activity of T-VEC inoculated into the respective lesion. ( E ) Box plots (with median, interquartile ranges, minimum, and maximum values) and ( F ) ROC curve analysis of responder and non-responder lesions with respect to Nectin-1, HVEM, STING, and cGAS immunostaining. p values for box plots were calculated using the Mann–Whitney test; p values for ROC curves analyzed the area under the curve.

Article Snippet: The following primary antibodies were used: Rabbit monoclonal antibody against STING (D2P2F, Cell Signaling; dilution for patient samples 1:50, dilution for cell lines 1:200), murine monoclonal antibody against Nectin-1 (R1.302.12, Santa Cruz, Heidelberg, Germany; dilution for patient samples 1:50), rabbit polyclonal antibody against Nectin-1 (AB_2736197, Invitrogen/Thermo Fisher; dilution for cell lines 1:100), murine monoclonal antibody against HVEM (CW10, Santa Cruz; dilution 1:500 for patient samples and cell lines), rabbit polyclonal antibody against cGAS (NBP1-86761, Novus Biologicals/Bio-Techne, Wiesbaden Nordenstadt, Germany; dilution for patient samples 1:200), and rabbit monoclonal antibody against cGAS (D1D3G) (Cell Signaling; dilution for cell lines 1:200).

Techniques: Injection, Immunostaining, Activity Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Rare longevity-associated variants, including a reduced-function mutation in cGAS, identified in multigenerational long-lived families

doi: 10.64898/2025.12.04.689698

Figure Lengend Snippet: A-B ) Quantitative PCR (qPCR) analysis of downstream gene expression ( A ) and western blot analysis of phosphorylated mediators ( B ) in the canonical cGAS-STING pathway in human mesenchymal stromal cells (hMSCs) expressing the longevity-associated cGAS variant (cGAS- rs200818241), wild-type cGAS (cGAS-WT), or an empty vector control (Ctrl). C ), qPCR analysis of downstream gene expression in the canonical cGAS-STING pathway in human granulosa (KGN) cells expressing cGAS-rs200818241, cGAS-WT, or Ctrl. D ), Western blot analysis of cGAS protein levels in fibroblasts (IMR-90) and primary human astrocytes expressing cGAS-rs200818241, cGAS-WT, or Ctrl. E ), Quantification of cGAS protein levels in hMSCs, normalized to GAPDH, showing reduced levels in cells expressing cGAS-rs200818241 compared to cGAS-WT. F ), Cycloheximide (CHX) chase assay showing faster degradation of the cGAS protein in hMSCs expressing cGAS-rs200818241 compared to cGAS-WT. All experiments were performed in triplicate. Statistical significance is denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The plasmid containing the cDNA of wild-type cGAS (pLenti-CMV-cGAS-HA) was obtained from Addgene.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Expressing, Variant Assay, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: Rare longevity-associated variants, including a reduced-function mutation in cGAS, identified in multigenerational long-lived families

doi: 10.64898/2025.12.04.689698

Figure Lengend Snippet: A ) Population doubling curves of cells expressing wild-type cGAS (cGAS-WT), the cGAS mutant (cGAS-rs200818241), or an empty vector control (Ctrl) in human mesenchymal stromal cells (hMSCs). Each dot indicates the cumulative population doubling per passage. cGAS-rs200818241 significantly extends cellular lifespan compared to cGAS-WT. B ) qPCR analysis of downstream genes in the canonical cGAS-STING signaling pathway and the senescence-associated p16 gene ( CDKN2A ). Expression of cGAS-rs200818241 increases both downstream genes in the canonical cGAS-STING pathway and p16 levels, while cGAS-rs200818241 reduces the expression of all these genes compared to cGAS-WT. C ), Senescence-associated β-galactosidase (SA-β-gal) staining assay showing that cGAS-WT increases the proportion of β-gal-positive cells, whereas cGAS-rs200818241 decreases the number of β-gal-positive cells compared to cGAS-WT. The left panel shows representative images of nuclei and SA-β-gal staining, the right panel presents the statistical analysis of β-gal-positive cells. All experiments were performed in triplicate. Statistical significance is denoted as follows: *p<0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The plasmid containing the cDNA of wild-type cGAS (pLenti-CMV-cGAS-HA) was obtained from Addgene.

Techniques: Expressing, Mutagenesis, Plasmid Preparation, Control, Staining

Journal: bioRxiv

Article Title: Rare longevity-associated variants, including a reduced-function mutation in cGAS, identified in multigenerational long-lived families

doi: 10.64898/2025.12.04.689698

Figure Lengend Snippet: A ) Significant reduced response of cGas in cGAS-rs200818241 mESCs compared to cGAS-WT mESCs upon stimulation by transfection for 24 hours. B ) Significant reduction of Sting in the cGAS-WT mESCs, but not the cGAS-rs200818241 mESCs, after stimulation by transfection for 24 hours. C ) Significant increase in phosphorylated Tbk1 (S172) over total Tbk1 in the cGAS-WT mESCs, but not the cGAS-rs200818241 mESCs, after stimulation by transfection for 24 hours. Vinculin was used for normalisation. The data shown is from two independent experiments with three technical replicates each. Data was analysed using a one-way ANOVA and Dunnett’s post hoc test. Statistical significance is denoted as follows: ns; not significant,*P < 0.05, **P < 0.01, *** p < 0.001.

Article Snippet: The plasmid containing the cDNA of wild-type cGAS (pLenti-CMV-cGAS-HA) was obtained from Addgene.

Techniques: Transfection

Journal: Journal for immunotherapy of cancer

Article Title: Hyperbaric oxygen facilitates teniposide-induced cGAS-STING activation to enhance the antitumor efficacy of PD-1 antibody in HCC.

doi: 10.1136/jitc-2021-004006

Figure Lengend Snippet: Figure 1 Teniposide triggered type Ⅰ interferon and NF-κB signaling through the cGAS-STING activation in human HCC cells. (A–C) Hep3B and Huh7 cells were treated with oxaliplatin or teniposide at each IC50 for 24 hours and the supernatant IFN-β was then measured by ELISA (A) and the mRNA expression of IFIT-1 and IFIT-2 (B) and CCL5 and CXCL10 (C) was measured by RT- qPCR. (D) Hep3B and Huh7 cells transduced with cGAS-shRNA or scramble-shRNA were treated with teniposide at each IC50 for 24 hours and the cellular protein expression of p-IRF3 and p-P65 was then detected by immunoblotting; β-actin was used as a loading control. (E) Hep3B and Huh7 cells transduced with STING-shRNA or scramble-shRNA were treated as in (D) and the cellular protein expression of p-IRF3 and p-P65 was then detected by immunoblotting. Data in (D) and (E) are representative of three independent experiments. Data in (A), (B) and (C) are shown as mean±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon genes; Ctrl, control; HCC, hepatocellular carcinoma; IC50, 50% inhibitory concentration; IFN-β, interferon β; IRF3, interferon regulatory factor 3; NF-κB, nuclear factor kappa-B; Oxa, oxaliplatin; Rel. expression, relative expression;RT-qPCR, real time quantitative PCR; sh-SCR, short hairpin RNA- scrambled; sh-cGAS, short hairpin RNA-cGAS; Teni, teniposide.

Article Snippet: The procedure for immunoblotting analyses was performed as previously described.21 The following antibodies were used for immunoblotting analyses: cGAS (Novus, NBP1- 86761), IRF3 (HUABIO, ET161214), P65 (CST, 6956), p- IRF3 (Abcam, ab76493), p- P65 (CST, 3033), HIF- 1α (CST, 14179), β-actin (Santa Cruz, sc- 47778), anti- mouse IgG (CST, 7076) and anti- rabbit IgG (CST, 7074).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Transduction, shRNA, Western Blot, Control, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: Journal for immunotherapy of cancer

Article Title: Hyperbaric oxygen facilitates teniposide-induced cGAS-STING activation to enhance the antitumor efficacy of PD-1 antibody in HCC.

doi: 10.1136/jitc-2021-004006

Figure Lengend Snippet: Figure 2 Hypoxia inhibited teniposide-induced cGAS-STING activation in human HCC cells. (A) Hep3B and Huh7 cells were cultured under a normoxic (21% O2) or a hypoxic (1% O2) condition for 18 hours and the cellular protein expression of HIF- 1α and cGAS was then detected by immunoblotting; β-actin was used as a loading control. (B) Hep3B and Huh7 cells were transfected with HT-DNA (5 µg/mL) and the cells were then cultured under either normoxic or hypoxic condition for 24 hours; the cellular protein expression of p-IRF3 was detected by immunoblotting. (C) Hep3B and Huh7 cells were treated with teniposide at each IC50, followed by either normoxic or hypoxic culture for 24 hours, and the cellular protein expression of p-IRF3 and p-P65 was then detected by immunoblotting. (D) Hep3B and Huh7 cells were treated as in (C) and the supernatant IFN-β was then measured by ELISA. (E–F) Hep3B and Huh7 cells were treated as in (C) and the mRNA expression of IFIT-1 and IFIT-2 (E) and CCL5 and CXCL10 (F) was then measured by RT-qPCR. Data in (A), (B) and (C) are representative of three independent experiments. Data in (D), (E) and (F) are shown as mean±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon genes; HCC, hepatocellular carcinoma; HIF-1α, hypoxia inducible factor 1α; HT-DNA, herring testes-DNA; IC50, 50% inhibitory concentration; IFN-β, interferon β; IRF3, interferon regulatory factor 3; Rel. expression, relative expression; RT-qPCR, real time quantitative PCR; Teni, teniposide.

Article Snippet: The procedure for immunoblotting analyses was performed as previously described.21 The following antibodies were used for immunoblotting analyses: cGAS (Novus, NBP1- 86761), IRF3 (HUABIO, ET161214), P65 (CST, 6956), p- IRF3 (Abcam, ab76493), p- P65 (CST, 3033), HIF- 1α (CST, 14179), β-actin (Santa Cruz, sc- 47778), anti- mouse IgG (CST, 7076) and anti- rabbit IgG (CST, 7074).

Techniques: Activation Assay, Cell Culture, Expressing, Western Blot, Control, Transfection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: Journal for immunotherapy of cancer

Article Title: Hyperbaric oxygen facilitates teniposide-induced cGAS-STING activation to enhance the antitumor efficacy of PD-1 antibody in HCC.

doi: 10.1136/jitc-2021-004006

Figure Lengend Snippet: Figure 3 Reoxygenation restored teniposide-induced cGAS-STING activation in human HCC cells. (A) Hep3B and Huh7 cells were cultured under either a normoxic (21% O2) or a hypoxic (1% O2) condition for 18 hours and the cells were then transferred to normoxic (21% O2) culture for 1 hour, 2 hours, 4 hours and 8 hours, respectively; the cellular protein expression of HIF-1α and cGAS was detected by immunoblotting at indicated time points and β-actin was used as a loading control. (B–D) Hep3B and Huh7 cells were treated with teniposide at each IC50, followed by hypoxic culture for 18 hours, and the cells were then transferred to normoxic culture for another 6 hours, and the cellular protein expression of p-IRF3 and p-P65 was detected by immunoblotting (B), the supernatant IFN-β was measured by ELISA (C), and the mRNA expression of CCL5 and CXCL10 was measured by RT-qPCR (D). (E–G) Hep3B and Huh7 cells transduced with HIF1A-shRNA or scramble-shRNA were treated with teniposide at each IC50, followed by either normoxic or hypoxic culture for 24 hours and the cellular protein expression of p-IRF3 and p-P65 was then detected by immunoblotting (E), the supernatant IFN-β was measured by ELISA (F), and the mRNA expression of CCL5 and CXCL10 was measured by RT-qPCR (G). Data in (A), (B) and (E) are representative of three independent experiments. Data in (C), (D), (F) and (G) are shown as mean±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon genes; HCC, hepatocellular carcinoma; HIF-1α, hypoxia inducible factor 1α; IC50, 50% inhibitory concentration; IFN-β, interferon β; IRF3, interferon regulatory factor 3; Rel. expression, relative expression; RT-qPCR, real time quantitative PCR; sh-SCR, short hairpin RNA-scrambled; Teni, teniposide.

Article Snippet: The procedure for immunoblotting analyses was performed as previously described.21 The following antibodies were used for immunoblotting analyses: cGAS (Novus, NBP1- 86761), IRF3 (HUABIO, ET161214), P65 (CST, 6956), p- IRF3 (Abcam, ab76493), p- P65 (CST, 3033), HIF- 1α (CST, 14179), β-actin (Santa Cruz, sc- 47778), anti- mouse IgG (CST, 7076) and anti- rabbit IgG (CST, 7074).

Techniques: Activation Assay, Cell Culture, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Transduction, shRNA, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: Journal for immunotherapy of cancer

Article Title: Hyperbaric oxygen facilitates teniposide-induced cGAS-STING activation to enhance the antitumor efficacy of PD-1 antibody in HCC.

doi: 10.1136/jitc-2021-004006

Figure Lengend Snippet: Figure 4 HBO facilitated teniposide to induce cGAS-STING signaling in mouse HCC tumor model. (A) Schematic illustration of the tumor microenvironment survey experiments in mouse HCC tumor model. Hepa1-6 liver orthotopically tumor-bearing mice were intraperitoneally injected with DMSO (Ctrl), oxaliplatin (10 mg/kg) and teniposide (10 mg/kg) twice at indicated time points. HBO therapy was administrated alone or after chemotherapy for a total of five times (n=3 per group). Tumors were resected and weighed after the final HBO administration. (B–C) HIF-1α expression of mice tumor tissues was determined by immunohistochemical staining (B) and immunoblotting (C) after HBO administration. Scale bars: 50 µm. (D) p-IRF3 expression of mice tumor tissues from different treatment groups was determined by immunohistochemical staining. Scale bars: 50 µm. (E) Ifn-b, Ccl5 and Cxcl10 mRNA expression of mice tumor tissues from different treatment groups was detected by RT-qPCR. (F) Representative images and quantitative analysis of p-IRF3low (n=60) and p-IRF3high (n=60) staining in tumor tissues from 120 patients with HCC. Scale bars: 50 µm (upper) and 25 µm (lower). (G) Disease-free survival (left) and overall survival (right) analyses of 120 patients with HCC showed a positive correlation between high p-IRF3 expression level and favorable prognosis. The prognostic significance was assessed by Cox regression analysis. (H–I) Kaplan–Meier curves were generated from the public microarray databases of human patients using an online software (kmplot.com/analysis/). Low expression levels of HIF1A (H) and high expression levels of IFN-B1, CCL5 and CXCL10 (I) were positively correlated with better survival of patients with HCC. Data in (C) are representative of three independent experiments. Data in (E) are shown as mean±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon genes; Ctrl, control; HBO, hyperbaric oxygen; DMSO, Dimethyl sulfoxide; HCC, hepatocellular carcinoma; HIF-1α, hypoxia inducible factor 1α; IRF3, interferon regulatory factor 3; Oxa, oxaliplatin; Rel. expression, relative expression; RT-qPCR, real time quantitative PCR; Teni, teniposide.

Article Snippet: The procedure for immunoblotting analyses was performed as previously described.21 The following antibodies were used for immunoblotting analyses: cGAS (Novus, NBP1- 86761), IRF3 (HUABIO, ET161214), P65 (CST, 6956), p- IRF3 (Abcam, ab76493), p- P65 (CST, 3033), HIF- 1α (CST, 14179), β-actin (Santa Cruz, sc- 47778), anti- mouse IgG (CST, 7076) and anti- rabbit IgG (CST, 7074).

Techniques: Injection, Expressing, Immunohistochemical staining, Staining, Western Blot, Quantitative RT-PCR, Generated, Microarray, Software, Control, Real-time Polymerase Chain Reaction

Journal: Journal for immunotherapy of cancer

Article Title: Hyperbaric oxygen facilitates teniposide-induced cGAS-STING activation to enhance the antitumor efficacy of PD-1 antibody in HCC.

doi: 10.1136/jitc-2021-004006

Figure Lengend Snippet: Figure 6 Combined HBO with teniposide chemotherapy sensitized tumor response to PD-1 antibody. (A) Schematic illustration of anti-PD-1 sensitization experiments in vivo. Mice with subcutaneously established tumors were intraperitoneally injected with DMSO (Ctrl) and teniposide (10 mg/kg) twice at indicated time points, and HBO therapy was administrated after teniposide chemotherapy in the HBO+teniposide+PD-1 combination group for a total of five times. PD-1 antibody (200 µg per mouse) was intraperitoneally injected every 3 days at indicated time points (n=5 per group). (B–D) Tumor growth curves of Hepa1-6 (B), MC38 (C) and B16 (D) bearing mice during anti-PD-1 sensitization experiments. (E) Schematic diagram of the combination therapy based on HBO and teniposide. *P<0.05, **P<0.01, ***P<0.001. cGAS, cyclic GMP-AMP synthase; CTL, cytotoxic T lymphocyte; Ctrl, control; DC, dendritic cell; DMSO, Dimethyl sulfoxide; HBO, hyperbaric oxygen; IFN-I, interferon type I; IFN-γ, interferon γ; IRF3, interferon regulatory factor 3; NF-κB, nuclear factor kappa-B; PD1, programmed cell death protein-1; STING, stimulator of interferon genes; Teni, teniposide; TNF-α, tumor necrosis factor-α.

Article Snippet: The procedure for immunoblotting analyses was performed as previously described.21 The following antibodies were used for immunoblotting analyses: cGAS (Novus, NBP1- 86761), IRF3 (HUABIO, ET161214), P65 (CST, 6956), p- IRF3 (Abcam, ab76493), p- P65 (CST, 3033), HIF- 1α (CST, 14179), β-actin (Santa Cruz, sc- 47778), anti- mouse IgG (CST, 7076) and anti- rabbit IgG (CST, 7074).

Techniques: In Vivo, Injection, Control

Journal: Journal for immunotherapy of cancer

Article Title: Hyperbaric oxygen facilitates teniposide-induced cGAS-STING activation to enhance the antitumor efficacy of PD-1 antibody in HCC.

doi: 10.1136/jitc-2021-004006

Figure Lengend Snippet: Figure 7 HBO boosted teniposide-induced cGAS-STING activation in surgically resected human HCC tumor tissues. (A) Representative immunofluorescence staining images of HIF-1α, p-IRF3 and DAPI in clinical tumor samples resected from patient 1 with HCC. Scale bar: 50 µm. (B, D, F) Clinical tumor samples were treated as in (A) and the tissular protein expression of HIF-1α, p-IRF3 and p-P65 from patient 1 (B), patient 2 (D) and patient 3 (F) was then detected by immunoblotting. (C, E, G) Clinical tumor samples were treated as in (A) and the tissular mRNA expression of IFNB1, IFIT-1, IFIT-2, CCL5 and CXCL10 from patient 1 (C), patient 2 (E) and patient 3 (G) was then measured by RT-qPCR. Data in (B), (D) and (F) are representative of three independent experiments. Data in (C), (E) and (G) are shown as mean±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon genes; DAPI, 4′,6-diamidino-2- phenylindole; DMSO, Dimethyl sulfoxide; HBO, hyperbaric oxygen; HCC, hepatocellular carcinoma; HIF-1α, hypoxia inducible factor 1α; IRF3, interferon regulatory factor 3; Rel. expression, relative expression; RT-qPCR, real time quantitative PCR; Teni, teniposide.

Article Snippet: The procedure for immunoblotting analyses was performed as previously described.21 The following antibodies were used for immunoblotting analyses: cGAS (Novus, NBP1- 86761), IRF3 (HUABIO, ET161214), P65 (CST, 6956), p- IRF3 (Abcam, ab76493), p- P65 (CST, 3033), HIF- 1α (CST, 14179), β-actin (Santa Cruz, sc- 47778), anti- mouse IgG (CST, 7076) and anti- rabbit IgG (CST, 7074).

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: cGAS-STING/HMGB1-mediated senescence induced by LRRK2 accelerates cartilage degeneration in osteoarthritis

doi: 10.1038/s41419-026-08651-y

Figure Lengend Snippet: A GEO analysis of significantly differentially expressed genes in OA patients and highlighted LRRK2 indicated by volcano plot. B The heatmap showed the gene expression patterns of NC versus OA humans. C The heat map showed significantly differentially expressed genes, in which LRRK2 was highlighted. D GO analysis of factors highly expressed in OA patients and LRRK2 was highlighted. E KEGG enrichment analysis was used to analyze the pathways related to LRRK2. F , G Immunohistochemical results of cartilage and synovial cells in rats, histogram showed that the OA group was significantly higher than the sham group. H Carrying out immunohistochemical detection and statistical analysis on the normal and OA articular cartilage of human knees. I Successful transfection was demonstrated by fluorescent expression of chondrocytes after LRRK2 transfection using lentivirus. J Western blotting detection and statistical analysis on overexpression and knockdown of LRRK2. K Cluster analysis of the samples showed a high degree of similarity, which confirmed the correctness of the experimental design and sample sampling. L The heatmap showed that the gene expression patterns and clustering relationships of the samples were similar. M The enrichment heatmap of cellular senescence pathways showed that many genes, including STING, cGAS, BCL2, P53, and Cdkn1a, were closely related to cell senescence. N Comparison between samples showed the number of differentially expressed genes. O GO enrichment analysis was used to analyze the function of related proteins after LRRK2 overexpression. P KEGG enrichment analysis was used to analyze the senescence-related pathways after LRRK2 overexpression. Q The connections among cGAS, STING, and HMGB1 in the protein interaction network was analyzed. R The predicted model diagram of molecular docking between LRRK2 and cGAS, along with the amino acids at the hydrogen bond binding sites. S The Co-IP method was used to detect the binding situation between LRRK2 and cGAS. T The Co-IP experiment was conducted on the control group and the IL-1β group. Under the senescence condition, the binding of LRRK2 to cGAS increased. U GSEA enrichment analysis of senescence-related gene expression. Data were presented as mean ± SD ( n = 3). Statistical analysis was performed using Student’s t test, *** p < 0.001.

Article Snippet: Anti-P53 (#21891-1-ap), TOM20 (#11802-1-ap), SOD2 (#24127-1-ap), MMP-3 (#17873-1-ap), Bcell lymphoma-2 (Bcl-2) (#26593-1-ap), TMEM173/STING (#19851-1-ap), P21 (#10355-1-ap), cGAS (#26416-1-ap), IL-6 (#21865-1-ap), TNF-α (#29652-1-ap), Vimentin (#10366-1-ap), and P16INK4A (#10883-1-ap) were purchased from Proteintech Group, Inc (Wuhan, China). β-tubulin (#T0023) was obtained from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Gene Expression, Immunohistochemical staining, Transfection, Expressing, Western Blot, Over Expression, Knockdown, Sampling, Comparison, Binding Assay, Co-Immunoprecipitation Assay, Control

Journal: Cell Death & Disease

Article Title: cGAS-STING/HMGB1-mediated senescence induced by LRRK2 accelerates cartilage degeneration in osteoarthritis

doi: 10.1038/s41419-026-08651-y

Figure Lengend Snippet: A , B The immunofluorescence of cGAS, STING, MMP-3, and P16 were analyzed in the articular cartilage of OA rats induced by DMM receiving rAAV-OE-LRRK2. C , D Immunohistochemical detection of the expressions of HMGB1 and P16 in the knee joints of rats. E , F The cGAS, STING, P16, and MMP-3 proportions in articular cartilage were quantified by immunofluorescence. G , H Statistical quantitative analysis of immunohistochemical staining in P16 and HMGB1. Data were expressed as mean ± SD ( n = 5). Statistical analysis was performed using Student’s t test, ** p < 0.01; *** p < 0.001.

Article Snippet: Anti-P53 (#21891-1-ap), TOM20 (#11802-1-ap), SOD2 (#24127-1-ap), MMP-3 (#17873-1-ap), Bcell lymphoma-2 (Bcl-2) (#26593-1-ap), TMEM173/STING (#19851-1-ap), P21 (#10355-1-ap), cGAS (#26416-1-ap), IL-6 (#21865-1-ap), TNF-α (#29652-1-ap), Vimentin (#10366-1-ap), and P16INK4A (#10883-1-ap) were purchased from Proteintech Group, Inc (Wuhan, China). β-tubulin (#T0023) was obtained from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Immunofluorescence, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: cGAS-STING/HMGB1-mediated senescence induced by LRRK2 accelerates cartilage degeneration in osteoarthritis

doi: 10.1038/s41419-026-08651-y

Figure Lengend Snippet: A The protein expressions of P53, P21, P16, MMP-3, BCL2, SOD2, TOM20, STING, cGAS, and HMGB1 were assessed via western blotting with β-tubulin as a loading control. B – E Quantitative analysis of relative protein expression. F The protein expressions of P53, P21, P16, MMP-3, BCL2, SOD2, TOM20, TNF-α, IL-6, and HMGB1 were assessed using the GTPase activity inhibitor ML141 via western blotting. β-tubulin was used as the internal normalization processing. G – J Quantitative analysis of relative protein expression ( n = 3). K Chondrocytes were stained by immunofluorescence with cGAS antibody and visualized using CY3 channels. L Chondrocytes were immunofluorescence stained with HMGB1antibody and visualized using CY3 channels. M Chondrocytes were immunofluorescence stained with STING antibody and visualized using FITC channels. N Chondrocytes were immunofluorescence stained with MMP-3 antibody and visualized using CY3 channels. O Quantitative analysis of cGAS immunofluorescence. P Quantitative analysis of STING immunofluorescence. Q The quantitative analysis of HMGB1 immunofluorescence. R Quantitative analysis of MMP-3 immunofluorescence. S Quantitative analysis of cell viability by CCK-8 assay. Data are presented as mean ± SD ( n = 5). Statistical analysis was performed using Student’s t test, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Anti-P53 (#21891-1-ap), TOM20 (#11802-1-ap), SOD2 (#24127-1-ap), MMP-3 (#17873-1-ap), Bcell lymphoma-2 (Bcl-2) (#26593-1-ap), TMEM173/STING (#19851-1-ap), P21 (#10355-1-ap), cGAS (#26416-1-ap), IL-6 (#21865-1-ap), TNF-α (#29652-1-ap), Vimentin (#10366-1-ap), and P16INK4A (#10883-1-ap) were purchased from Proteintech Group, Inc (Wuhan, China). β-tubulin (#T0023) was obtained from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Western Blot, Control, Expressing, Activity Assay, Staining, Immunofluorescence, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: cGAS-STING/HMGB1-mediated senescence induced by LRRK2 accelerates cartilage degeneration in osteoarthritis

doi: 10.1038/s41419-026-08651-y

Figure Lengend Snippet: A The protein expressions of P53, P21, P16, MMP-3, BCL2, SOD2, TOM20, TNF-α, IL-6, STING, cGAS, and HMGB1 were assessed via western blotting. β-tubulin was used as the internal normalization processing. B – E Quantitative analysis of relative protein expression ( n = 3). F The protein expressions of P53, P21, P16, MMP-3, BCL2, SOD2, TOM20, TNF-α, IL-6, STING, cGAS, and HMGB1 were assessed with LRRK2 silence via western blotting. β-tubulin was used as the internal normalization processing. G – J Quantitative analysis of relative protein expression ( n = 3). K Immunofluorescence double-labeling analysis of cGAS and STING proteins. L Immunofluorescence analysis of HMGB1 expression and localization. M Immunofluorescence detection of MMP-3 and TNF-α expression. N Statistical analysis of cGAS and STING protein expression ( n = 5). O Statistical analysis of HMGB1 protein expression ( n = 5). P Statistical analysis of MMP-3 and TNF-α protein expression ( n = 5). Statistical analysis was performed using Student’s t test, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Anti-P53 (#21891-1-ap), TOM20 (#11802-1-ap), SOD2 (#24127-1-ap), MMP-3 (#17873-1-ap), Bcell lymphoma-2 (Bcl-2) (#26593-1-ap), TMEM173/STING (#19851-1-ap), P21 (#10355-1-ap), cGAS (#26416-1-ap), IL-6 (#21865-1-ap), TNF-α (#29652-1-ap), Vimentin (#10366-1-ap), and P16INK4A (#10883-1-ap) were purchased from Proteintech Group, Inc (Wuhan, China). β-tubulin (#T0023) was obtained from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Labeling

Journal: Nature Communications

Article Title: DNA methylation protects cancer cells against senescence

doi: 10.1038/s41467-025-61157-7

Figure Lengend Snippet: A RT-qPCR on cGAS in HCT116 lines upon Dox/Auxin treatment. N = 3 biological replicates. B Western blotting of cGAS protein in HCT116 lines at the indicated days upon Dox/Auxin treatment. C Scheme of cGAS knockdown experiments. D Total cell numbers at the indicated days upon Dox/Auxin treatment. N = 3 technical replicates. E SA-β-gal staining (left panel) and quantification (right panel) of the indicated lines. N = 5 fields of view. F RT-qPCR results of cGAS and selected SASP genes. G Scheme of STING knockout experiments. H SA-β-gal staining (left panel) and quantification (right panel). N = 5 fields of view. I RT-qPCR results of selected SASP genes. J Western blotting of cGAS in cytoplasmic and nuclear fractions of HCT116 lines at the indicated days upon Dox/Auxin treatment. All scale bars are 50 µm. Data of A , D , E , H are presented as mean ± SD. Data of A are analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. Data of D , E , H are analyzed by two-way ANOVA with Sidak’s multiple comparisons test. Heatmap data of F , I are presented as mean from three technical replicates. In all figures, we use the following convention: ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: non-significant. Source data are provided as a file.

Article Snippet: The wildtype cGAS (Addgene #102612) and NLS- cGAS (Addgene #127656) overexpression plasmids were also obtained from Nicolas Manel.

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Staining, Knock-Out

Journal: Scientific Reports

Article Title: CMPK2 promotes microglial activation through the cGAS-STING pathway in the neuroinflammatory mechanism

doi: 10.1038/s41598-025-97232-8

Figure Lengend Snippet: LPS Enhances microglial-mediated neuroinflammation via the cGAS/STING Pathway. ( A ) Western blot analysis of cGAS and STING protein levels under different treatments. ( B ) Analysis of cGAS and STING mRNA expression under different treatments. ( C ) Flow cytometry analysis of oxidative stress (ROS) levels under different treatments. D , E . Analysis of mRNA and protein expression levels of IL-6, TNF-α, IL-1β, and TGF-β. The presented data represent the mean ± standard deviation (SD) derived from three independent experiments. Statistical significance of the fold change was determined, with * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 denoting the significance levels.

Article Snippet: To overexpress/knockdown CMPK2 and knockdown cGAS in BV2 cells, we obtained the CMPK2 (NM_207315) Human Tagged ORF clone (RC218794), CMPK2 Human siRNA Oligo Duplex (SR315070), and Human cGAS shRNA Plasmid Kit (TL305813) from OriGene Technologies (Rockville).

Techniques: Western Blot, Expressing, Flow Cytometry, Standard Deviation, Derivative Assay

Journal: Scientific Reports

Article Title: CMPK2 promotes microglial activation through the cGAS-STING pathway in the neuroinflammatory mechanism

doi: 10.1038/s41598-025-97232-8

Figure Lengend Snippet: Inhibition of the cGAS/STING Pathway Alleviates microglial-mediated neuroinflammation. ( A ) Western blot analysis of cGAS and STING protein levels under different treatments. ( B ) Analysis of cGAS and STING mRNA expression under different treatments. ( C ) Immunofluorescence analysis of cGAS and STING expression in mouse primary microglial under different treatments. ( D ) Flow cytometry analysis of oxidative stress (ROS) levels under different treatments. E, F. Analysis of mRNA and protein expression levels of IL-6, TNF-α, IL-1β, and TGF-β. The presented data represent the mean ± standard deviation (SD) derived from three independent experiments. Statistical significance of the fold change was determined, with * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 denoting the significance levels.

Article Snippet: To overexpress/knockdown CMPK2 and knockdown cGAS in BV2 cells, we obtained the CMPK2 (NM_207315) Human Tagged ORF clone (RC218794), CMPK2 Human siRNA Oligo Duplex (SR315070), and Human cGAS shRNA Plasmid Kit (TL305813) from OriGene Technologies (Rockville).

Techniques: Inhibition, Western Blot, Expressing, Immunofluorescence, Flow Cytometry, Standard Deviation, Derivative Assay

Journal: Scientific Reports

Article Title: CMPK2 promotes microglial activation through the cGAS-STING pathway in the neuroinflammatory mechanism

doi: 10.1038/s41598-025-97232-8

Figure Lengend Snippet: CMPK2 Induces microglial-mediated neuroinflammation via the cGAS/STING Pathway. A . Western blot analysis of CMPK2 under control plasmid and CMPK2 overexpression plasmid groups. B .Western blot analysis of CMPK2, cGAS, and STING protein levels under different treatments. C . Analysis of CMPK2, cGAS, and STING mRNA expression under different treatments. D . Analysis of morphological differences between treated and control cells. E . Analysis of microglial activation marker CD40 expression. F . Molecular docking analysis of CMPK2 and cGAS interaction. G , H . Analysis of mRNA and protein expression levels of IL-6, TNF-α, IL-1β, and TGF-β. The presented data represent the mean ± standard deviation (SD) derived from three independent experiments. Statistical significance of the fold change was determined, with * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 denoting the significance levels.

Article Snippet: To overexpress/knockdown CMPK2 and knockdown cGAS in BV2 cells, we obtained the CMPK2 (NM_207315) Human Tagged ORF clone (RC218794), CMPK2 Human siRNA Oligo Duplex (SR315070), and Human cGAS shRNA Plasmid Kit (TL305813) from OriGene Technologies (Rockville).

Techniques: Western Blot, Control, Plasmid Preparation, Over Expression, Expressing, Activation Assay, Marker, Standard Deviation, Derivative Assay