Journal: Scientific Reports

Article Title: MC4R and ENPP1 gene polymorphisms and their implication in maternal and neonatal risk for obesity

doi: 10.1038/s41598-019-47402-2

Figure Lengend Snippet: Associations between MC4R rs17782313 and ENPP1 rs1044498 SNPs and GWG status.

Article Snippet: In this respect we used the following single tub TaqMan SNP Genotyping assay formats: C__32667060_10 for rs17782313, and C__1207994_20 for rs1044498 (from ThermoFisher Scientific).

Techniques:

Journal: Scientific Reports

Article Title: MC4R and ENPP1 gene polymorphisms and their implication in maternal and neonatal risk for obesity

doi: 10.1038/s41598-019-47402-2

Figure Lengend Snippet: Relationship between studied gene polymorphisms and neonatal anthropometric bioimpedance characteristics.

Article Snippet: In this respect we used the following single tub TaqMan SNP Genotyping assay formats: C__32667060_10 for rs17782313, and C__1207994_20 for rs1044498 (from ThermoFisher Scientific).

Techniques:

Journal: iScience

Article Title: CD38 restrains the activity of extracellular cGAMP in a model of multiple myeloma

doi: 10.1016/j.isci.2024.109814

Figure Lengend Snippet:

Article Snippet: The Probes used were the following: GUSB : Hs99999909_m1; IFNA1 : Hs03044218_g1; IFNA2 : Hs00265051_s1; IFNA4 : Hs01681284_sH; IFNB1 : Hs01077958_s1; IFNE : Hs00703565_s1; IFNW1 : Hs00958789_s1; IFNL1 : Hs00601677_g1; IFI6 : Hs00242571_m1; IFIH1 : Hs00223420_m1; IFIT1 : Hs03027069_s1; IFIT2 : Hs01584837_s1; IFIT3 : Hs01922752_s1; IFITM1 : Hs00705137_s1; IRF7 : Hs01014809_g1; ISG15 : Hs01921425_s1; ISG20 : Hs00158122_m1; IFI27 : Hs01086373_g1; IFI44L : Hs00915292_m1; RSAD2 : Hs00369813_m1; DDX58 (RIG-I): Hs01061436_m1; TGFB1 : Hs00998133_m1; IL10 : Hs00961622_m1; IL15 : Hs01003716_m1; IL6 : Hs00174131_m1; IL1B : Hs01555410_m1; CCL5 : Hs00982282; CXCL9 : Hs00171065_m1; CXCL10 : Hs00171042_m1; CXCL11 : Hs00171138_m1; IFNG : Hs00989291_m1; ENPP1 : Hs01054040_m1; CD73 : Hs00159686_m1.

Techniques: Control, Virus, Isolation, Recombinant, Enzyme-linked Immunosorbent Assay, Protein Extraction, Labeling, Sequencing, Plasmid Preparation, Software, Staining

Journal: Archives of toxicology

Article Title: Cannabidivarin and cannabigerol induce unfolded protein response and angiogenesis dysregulation in placental trophoblast HTR-8/SVneo cells.

doi: 10.1007/s00204-024-03781-8

Figure Lengend Snippet: Fig. 2 CBDV and CBG increase the expression of ER-stress mark- ers. HTR-8/SVneo cells were treated with CBDV and CBG (5 μM) for 48 h, with or without the TRPV1 antagonist CPZ (0.2 µM). A-D Expression of the HSPA5, sXBP1, ATF4 and DDIT3 genes was analyzed through RT-PCR. Results show transcript levels normal- ized against ACTB. E The protein expression of p-eIF2α and CHOP

Article Snippet: Membranes were incubated with mouse monoclonal antibodies against CHOP (1:100, sc-7351; Santa Cruz Biotechnology, CA, USA) and TRB3 (1:100, sc-271572; Santa Cruz Biotechnology, CA, USA), or rabbit monoclonal or polyclonal antibodies against p-eIF2α (1:200, 3398S; Cell Signaling Technology, Leiden, The Netherlands), eIF2α (1:200, 5342S; Cell Signaling Technology, Leiden, The Netherlands), caspase-3 (1:200, sc-7148; Santa Cruz Biotechnology, CA, USA), poly (ADP-ribose) polymerase-1 (PARP-1) (1:200, 9542S; Cell Signaling Technology, Leiden, The Netherlands), p-AKT (Ser473) (1:200, 4060S; Cell Signaling Technology, Leiden, The Netherlands) and AKT (1:200, 4691S; Cell Signaling Technology, Leiden, The Netherlands) at 4 °C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Advanced Science

Article Title: T Cell‐Derived Apoptotic Extracellular Vesicles Hydrolyze cGAMP to Alleviate Radiation Enteritis via Surface Enzyme ENPP1

doi: 10.1002/advs.202401634

Figure Lengend Snippet: ApoEVs hydrolyze cGAMP by ENPP1 to inhibit the activation of the cGAS‐STING pathway. A) Western blot analysis of ENPP1 expression in naive T cells, activated T cells, apoptotic T cells, and ApoEVs. B) Immunoelectron microscopy detection of ENPP1 on ApoEVs. Scale bar, 200 nm. Arrows indicate ENPP1 adhered by gold particles. C) The activity of ApoEVs to hydrolyze cGAMP with or without ENPP1‐IN (ENPP1 inhibitor, 100 µ m ) was measured in vitro ( n = 3). DMSO was used as solvent control. D) Schematic diagram of the experimental procedure. To evaluate the effect of ENPP1 on ApoEVs surface on neutrophils and macrophages, ApoEVs treated with or without ENPP1‐IN were added to cGAMP‐pretreated neutrophils or BMDM. DMSO was used as solvent control. 6 h after ApoEVs were added, the cGAMP concentration, STING protein, and IFNB mRNA expression of cells were detected. E) The detection of extracellular and intracellular cGAMP concentration for neutrophils ( n = 3). F) Western blot analysis of STING protein in neutrophils. G) The mRNA expression of IFNB in neutrophils ( n = 3). H) The detection of extracellular and intracellular cGAMP concentration for BMDM ( n = 3). I) Western blot analysis of STING protein in BMDM. J) The mRNA expression of IFNB in BMDM ( n = 3). The data are represented as mean ± SD. Statistical analyses are performed by one‐way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, p > 0.05.

Article Snippet: Subsequently, 0, 25, 50, 100 µg mL −1 ApoEVs added with or without 100 µ m ENPP1 inhibitor (ENPP1‐IN‐12, MedChemExpress, HY‐143256) were reacted with 10 µ m cGAMP (MedChemExpress, HY‐100564A) in 100 µL of DMEM (free EVs) for 2 h at room temperature.

Techniques: Activation Assay, Western Blot, Expressing, Immuno-Electron Microscopy, Activity Assay, In Vitro, Solvent, Control, Concentration Assay

Journal: Advanced Science

Article Title: T Cell‐Derived Apoptotic Extracellular Vesicles Hydrolyze cGAMP to Alleviate Radiation Enteritis via Surface Enzyme ENPP1

doi: 10.1002/advs.202401634

Figure Lengend Snippet: ApoEVs hydrolyze intracellular cGAMP. A, B) The presence of ENPP1 on the surface of intracellular ApoEVs was observed after the uptake of ApoEVs by BMDM. To avoid the effects of ENPP1 on the cell itself, ApoEVs were incubated with ENPP1 antibodies before they were added to BMDM. A) Representative images of PKH26‐labeled ApoEVs (red) and ENPP1 (green) detected by laser scanning confocal microscopy. Scale bar, 50 µm in low‐magnification images and 10 µm in high‐magnification images. B) ENPP1 of intracellular ApoEVs detected by immunoelectron microscopy. Scale bar, 2 µm in low‐magnification images and 200 nm in high‐magnification images. C) Schematic diagram of the experimental procedure. To evaluate the ENPP1 enzymatic activity of intracellular ApoEVs in BMDM, ApoEVs treated with or without ENPP1‐IN (ENPP1 inhibitor, 100 µ m ) were added to BMDM. DMSO was used as solvent control. After ApoEVs were added, unengulfed ApoEVs were removed by changing the medium, and cells were stimulated with cGAMP for 5 h. Concurrently, by the addition of purified soluble recombinant mouse ENPP1, it was proved that the secretory ENPP1 could not hydrolyze intracellular cGAMP. D) Concentration of extracellular and intracellular cGAMP in BMDM. E) Western blot analysis of the STING expression. F) The mRNA expression of IFNB . The data are represented as mean ± SD. Statistical analyses are performed by one‐way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, p > 0.05.

Article Snippet: Subsequently, 0, 25, 50, 100 µg mL −1 ApoEVs added with or without 100 µ m ENPP1 inhibitor (ENPP1‐IN‐12, MedChemExpress, HY‐143256) were reacted with 10 µ m cGAMP (MedChemExpress, HY‐100564A) in 100 µL of DMEM (free EVs) for 2 h at room temperature.

Techniques: Incubation, Labeling, Confocal Microscopy, Immuno-Electron Microscopy, Activity Assay, Solvent, Control, Purification, Recombinant, Concentration Assay, Western Blot, Expressing

Journal: Advanced Science

Article Title: T Cell‐Derived Apoptotic Extracellular Vesicles Hydrolyze cGAMP to Alleviate Radiation Enteritis via Surface Enzyme ENPP1

doi: 10.1002/advs.202401634

Figure Lengend Snippet: ApoEV administration alleviates radiation enteritis by hydrolyzing cGAMP. A) Schematic diagram of the in vivo experimental procedure. Before ApoEVs injection, ApoEVs were incubated with ENPP1‐IN to block ENPP1 enzyme activity. The mice exposed to ionizing radiation (IR) were intravenously injected with PBS (150 µL), ApoEVs (150 µg in 150 µL PBS), or ENPP1‐IN incubated ApoEVs (150 µg in 150 µL PBS) on days 0.5, 2 and 4 after radiation. B) Survival rate of mice ( n = 10), significance tested using Log‐rank test. C) Body weight of mice ( n = 5–6). D) Representative morphology images of the colon and quantitative analysis of the colon length in each group ( n = 6). E) Representative H&E staining of the small intestine and colon tissues, and quantitative analysis of the histological activity index ( n = 6). Scale bar, 500 µm in low‐magnification images and 75 µm in high‐magnification images. F–H) cGAMP concentration ( n = 5, F), STING expression (G), and IFNB mRNA level (H) in the small intestine. I–K) cGAMP concentration ( n = 5) (I), STING expression (J), and IFNB mRNA level (K) in the colon. L) Serum concentrations of IFNβ, IL‐6, and IL‐10 detected by ELISA ( n = 6). The data are represented as mean ± SD. Statistical analyses are performed by one‐way ANOVA with Tukey's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, p > 0.05.

Article Snippet: Subsequently, 0, 25, 50, 100 µg mL −1 ApoEVs added with or without 100 µ m ENPP1 inhibitor (ENPP1‐IN‐12, MedChemExpress, HY‐143256) were reacted with 10 µ m cGAMP (MedChemExpress, HY‐100564A) in 100 µL of DMEM (free EVs) for 2 h at room temperature.

Techniques: In Vivo, Injection, Incubation, Blocking Assay, Activity Assay, Staining, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature chemical biology

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: ( a ) Ion dependency and ( b ) pH preference of the dominant hydrolase activity in MDA-MB231 cells. ( c ) Activity of recombinant ENPP1 alone or ( d ) coupled to alkaline phosphatase in buffer condition: 0.2% NP-40, 20 mM Tris-HCl, pH 9.0, 2 mM Ca 2+ , 200 μM Zn 2+ . ( e ) Kinetics of 2′3′-cGAMP and ATP hydrolysis by recombinant ENPP1. 1 nM ENPP1 was tested in the same buffer condition as ( f ) and ( g ). Data are presented as mean and standard error.

Article Snippet: Recombinant ENPP1 was purchased from R&D systems (6136-EN-010).

Techniques: Activity Assay, Recombinant

Journal: Nature chemical biology

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: ( a ) Knockdown of ENPP1 in MDA-MB231 cells diminished hydrolase activity. Four siRNA oligos against ENPP1 with a control siRNA sequence against PDE12 were used. 4 days after siRNA transfection, cells were lysed and assayed for activity (upper panel) and blotted for ENPP1 level (lower level). ( b ) Hydrolase activity in plasma from Enpp1 -/- mice and their littermates. ( c ) Western blot characterization of Enpp1 -/- mice. ( d ) Hydrolase activity in livers and spleens from Enpp1 -/- mice and their littermates. Livers and spleens were minced and Dounce homogenized in lysis buffer: 1% NP-40, 20 mM Tris-HCl, pH 7.5, protease inhibitor cocktail. The assay was conducted in 0.2% NP-40, 20 mM Tris-HCl, 150 mM KCl, 2 mM Ca 2+ , 2 mM Mg 2+ , 200 μM Zn 2+ at the indicated pH. NaOAc buffer was used for pH 5.0-6.0; PIPES buffer was used for pH 6.5 and 7.0 and 2′3′-cGAMP runs at a lower R f in this buffer; Tris-HCl buffer was used for pH 7.5-9.0, and Borate buffer was used pH 9.5. These buffer conditions were also used in liver and spleen extract studies.

Article Snippet: Recombinant ENPP1 was purchased from R&D systems (6136-EN-010).

Techniques: Knockdown, Activity Assay, Control, Sequencing, Transfection, Clinical Proteomics, Western Blot, Lysis, Protease Inhibitor

Journal: Nature chemical biology

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: ( a ) Scheme of enzymatic synthesis of 3′3′-cGAMP and 2′3′-cGAMP analogs. For 3′3′-cGAMP, 50 nM of DncV was incubated with 1 mM ATP and 1 mM GTP in 1 mL buffer containing 20 mM Tris-HCl, pH 8.0, 20 mM MgCl 2 for 3 h at room temperature. For 2′3′-cGAMP analogs, 1-10 μM mouse cGAS (amino acid residues 147-507) was incubated with 1 mM ATP, 1 mM GTP, and 0.1 mg/mL HT-DNA in 1 mL of the same buffer for 12 h at room temperature. ( b ) Hydrolysis reactions of ENPP1 (1 nM) with the analogs (10 μM). ( c ) Scintillation proximity assay to measure the binding affinity of the analogs towards hSTING (amino acid residues 139-379). Biotinylated hSTING (100 nM) was immobilized onto 96-well streptavidin coated SPA plates. Neat 35 S-labeled 2′3′-cGA s MP (500 pM) was used as the probe. N=3 samples. Data are presented as mean and standard error. ( d ) IFN-β production in THP-1 cells stimulated with the analogs. THP-1 cells were incubated with the analogs at the indicated concentrations for 24 h. IFN-β in the media was measured using a HEK-SEAP cell line. Representative data from a biological triplicate is depicted.

Article Snippet: Recombinant ENPP1 was purchased from R&D systems (6136-EN-010).

Techniques: Incubation, Scintillation Proximity Assay, Binding Assay, Labeling

Journal: Nature chemical biology

Article Title: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs

doi: 10.1038/nchembio.1661

Figure Lengend Snippet: Lung fibroblast cells from wild type and Enpp1 -/- female mice were incubated with 2′3′-cGAMP analogs and HSV-60 at the indicated concentrations for 24 h. IFN-β in the media was measured using a B16-SEAP cell line. N=3 samples. Data are presented as mean and standard error.

Article Snippet: Recombinant ENPP1 was purchased from R&D systems (6136-EN-010).

Techniques: Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Pyrophosphate therapy prevents trauma‐induced calcification in the mouse model of neurogenic heterotopic ossification

doi: 10.1111/jcmm.15793

Figure Lengend Snippet: Plasma pyrophosphate level and expression of genes Abcc6 and Enpp1 in the liver during trauma‐induced calcification. A: Plasma pyrophosphate level before (0) and at different time‐points after TBI + CTX. B and C: Expression of genes in the liver controlling plasma pyrophosphate level (B: Abcc6 , C: Enpp1 ). Expression levels were normalized to that of beta2microglobulin

Article Snippet: Expression level of Abcc6 (Mm00497698_m1) and Enpp1 (Mm01193761_m1) in liver and Runx2 (Mm00501584_m1), Sox9 (Mm00448840_m1), Bmp‐2 (Mm01340178_m1) and Bmp‐4 (Mm00432087_m1) in muscle was detected using commercially available TaqMan probes (Thermo Fisher Scientific, MA, USA).

Techniques: Clinical Proteomics, Expressing