Journal: International Journal of Nanomedicine

Article Title: T Cell-Derived Apoptotic Extracellular Vesicles Ameliorate Bone Loss via CD39 and CD73-Mediated ATP Hydrolysis

doi: 10.2147/IJN.S491222

Figure Lengend Snippet: ApoEVs hydrolyze ATP to adenosine via surface CD39 and CD73. ( A ) The activity of ApoEVs and apoptotic T cells to hydrolyze ATP was measured in vitro (N = 3). ( B ) Extracellular ATP concentration in bone marrow plasma of sham and OVX animals (N = 6). ( C ) Western blot analysis of CD39 and CD73 in bone marrow of sham and OVX animals. ( D ) Extracellular adenosine concentration in bone marrow plasma of sham and OVX animals. ( E ) Western blot analysis of CD39 and CD73 on the membrane of apoptotic T cells and ApoEVs. ( F ) Immunoelectron microscopy detection of CD39 and CD73 on ApoEVs (scale bar = 200 nm). Yellow arrows indicate CD39 and red arrows indicate CD73 adhered by gold particles. ( G ) The activity of ApoEVs to hydrolyze ATP with or without POM (a CD39 inhibitor) or PSB (a CD73 inhibitor) was determined in vitro (N = 3). ( H ) The activity of ApoEVs in hydrolyzing ATP to adenosine with or without POM or PSB was determined in vitro (N = 3). Data are presented as mean ± SD; ns, not significant; ***P< 0.001 by one-way ANOVA with Tukey’s post hoc test or unpaired Student’s t test.

Article Snippet: To prepare CD39- or CD73-inhibited ApoEVs, 30 μg/mL of ApoEVs were preincubated with 100 μM POM (dissolved in DMSO, MedChemExpress) or PSB (dissolved in water, MedChemExpress) for 40 minutes at room temperature.

Techniques: Activity Assay, In Vitro, Concentration Assay, Clinical Proteomics, Western Blot, Membrane, Immuno-Electron Microscopy

Journal: International Journal of Nanomedicine

Article Title: T Cell-Derived Apoptotic Extracellular Vesicles Ameliorate Bone Loss via CD39 and CD73-Mediated ATP Hydrolysis

doi: 10.2147/IJN.S491222

Figure Lengend Snippet: ApoEVs promote bone regeneration via surface CD39 and CD73. OVX mice were divided into four groups and injected with PBS, ApoEVs, and POM or PSB pretreated ApoEVs, respectively (N = 5–6). Extracellular ATP ( A ) and adenosine ( B ) concentration in bone marrow plasma in different groups of mice were detected. ( C ) Micro-CT analyses of trabecular bone mass in the femurs. ( D-G ) Quantitative analyses of BMD ( D ), BV/TV ( E ), Tb. N ( F ) and Tb. Sp ( G ). ( H-J ) ELISA assays of IFN-γ ( H ), IL-17 ( I) and TNF-α ( J ) concentrations in the serum of peripheral blood from indicated groups. Data are presented as mean ± SD; ns, not significant; *P< 0.05; **P< 0.01; ***P< 0.001 by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: To prepare CD39- or CD73-inhibited ApoEVs, 30 μg/mL of ApoEVs were preincubated with 100 μM POM (dissolved in DMSO, MedChemExpress) or PSB (dissolved in water, MedChemExpress) for 40 minutes at room temperature.

Techniques: Injection, Concentration Assay, Clinical Proteomics, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Technology in Cancer Research & Treatment

Article Title: B7-H3 and CD39 Co-Localization in Gastric Cancer: A Potential Prognostic Biomarker and Potential Dual-Target for Immunotherapy

doi: 10.1177/15330338251380957

Figure Lengend Snippet: Expression Patterns of B7-H3 and CD39 in Gastric Precancerous Lesions and Gastric Cancer Tissues. (A) Representative IHC Expression Patterns of B7-H3 and CD39 Across Different Pathological Stages (CSG, CAG, LGIN, HGIN, and GC). (B) Quantitative Analysis of Positive Expression Areas of CD39. (C) Quantitative Analysis of Positive Expression Areas of B7-H3. (D) Representative Multiplex IHC Expression Patterns of B7-H3 and CD39 Across Different Pathological Stages (CSG, CAG, LGIN, HGIN, and GC). (E) Quantitative Analysis of Multiplex IHC Expression of B7-H3 and CD39. (F) Representative Co-Localization Expression of B7-H3 and CD39 in GC. IHC Scale Bars: 50 µm; mIHC Scale Bars: 100 µm.

Article Snippet: The following primary antibodies were used according to the manufacturer's instructions: mouse anti-human B7-H3 monoclonal antibody, 1/200 dilution (Cat No: 66 481-1-Ig, Proteintech, China), rabbit anti-human CD39 polyclonal antibody, 1/1000 dilution (Cat No: 14211-1-AP, Proteintech, China) and mouse anti-human CD8 monoclonal antibody, 1/10 000 dilution (Cat No: 66868-1-Ig, Proteintech, China).

Techniques: Expressing, Multiplex Assay

Journal: Technology in Cancer Research & Treatment

Article Title: B7-H3 and CD39 Co-Localization in Gastric Cancer: A Potential Prognostic Biomarker and Potential Dual-Target for Immunotherapy

doi: 10.1177/15330338251380957

Figure Lengend Snippet: Co-Localization of B7-H3 and CD39 in Gastric Cancer Cells Indicates Poor Prognosis. (A, B) Representative Immunohistochemical Images Showing Low and High Expression of B7-H3 (A) and CD39 (B) in Gastric Cancer (GC) Specimens. (C) Correlation Analysis of B7-H3 and CD39 Expression. (F, G, H, I) Kaplan-Meier Survival Curves for Overall Survival of GC Patients Based on the Expression Status of B7-H3 (F), CD39 (G), Dual High Expression of B7-H3 and CD39 (H), and Co-Localized Expression Status of B7-H3-CD39 (I). Scale Bars: 100 µm.

Article Snippet: The following primary antibodies were used according to the manufacturer's instructions: mouse anti-human B7-H3 monoclonal antibody, 1/200 dilution (Cat No: 66 481-1-Ig, Proteintech, China), rabbit anti-human CD39 polyclonal antibody, 1/1000 dilution (Cat No: 14211-1-AP, Proteintech, China) and mouse anti-human CD8 monoclonal antibody, 1/10 000 dilution (Cat No: 66868-1-Ig, Proteintech, China).

Techniques: Immunohistochemical staining, Expressing

Journal: Technology in Cancer Research & Treatment

Article Title: B7-H3 and CD39 Co-Localization in Gastric Cancer: A Potential Prognostic Biomarker and Potential Dual-Target for Immunotherapy

doi: 10.1177/15330338251380957

Figure Lengend Snippet: Absence of Correlation Between Co-localization of B7-H3 and CD39 Expression and CD8 + T Cell Infiltration in Gastric Cancer. (A, B) Representative Immunohistochemical Images Showing the Expression of B7-H3 with CD8 (A) or CD39 with CD8 (B) in the Same Patient. (C) Correlation Between B7-H3 Expression and CD8 Expression, and Between CD39 Expression and CD8 Expression. (D, E) Representative Multiplex Immunohistochemistry Images of B7-H3, CD39, and CD8 (D), and Correlation Analysis Between the Co-Localization Expression Score of B7-H3 and CD39 and the Extent of CD8 Infiltration (E). (F, G) Representative Multiplex Immunohistochemistry Images of CD39 and CD8, with Arrows Indicating CD39 + CD8 + T Cells (F), and the Proportion of CD39 + CD8 + T Cells within the CD8 + T Cell Population (G). Scale Bars: 100 µm.

Article Snippet: The following primary antibodies were used according to the manufacturer's instructions: mouse anti-human B7-H3 monoclonal antibody, 1/200 dilution (Cat No: 66 481-1-Ig, Proteintech, China), rabbit anti-human CD39 polyclonal antibody, 1/1000 dilution (Cat No: 14211-1-AP, Proteintech, China) and mouse anti-human CD8 monoclonal antibody, 1/10 000 dilution (Cat No: 66868-1-Ig, Proteintech, China).

Techniques: Expressing, Immunohistochemical staining, Multiplex Assay, Immunohistochemistry

Journal: Technology in Cancer Research & Treatment

Article Title: B7-H3 and CD39 Co-Localization in Gastric Cancer: A Potential Prognostic Biomarker and Potential Dual-Target for Immunotherapy

doi: 10.1177/15330338251380957

Figure Lengend Snippet: Kaplan-Meier Survival Curves for Gastric Cancer Patients Based on Specific Expression Statuses and Immune Cell Infiltration Levels. (A, B, C, D) Kaplan-Meier Survival Curves for GC Patients Stratified by B7-H3 High CD8 Low (A) Expression Status 、 CD39 High CD8 Low (B) Expression Status 、 B7-H3-CD39 Both high CD8 Low Expression Status (C) and B7-H3-CD39 Co-localization CD8 low Expression Status (D). (E, F) Kaplan-Meier Survival Curves for GC Patients Based on CD8 + T Cell Infiltration Levels (E) and CD39 + CD8 + T Cell Infiltration Status (F).

Article Snippet: The following primary antibodies were used according to the manufacturer's instructions: mouse anti-human B7-H3 monoclonal antibody, 1/200 dilution (Cat No: 66 481-1-Ig, Proteintech, China), rabbit anti-human CD39 polyclonal antibody, 1/1000 dilution (Cat No: 14211-1-AP, Proteintech, China) and mouse anti-human CD8 monoclonal antibody, 1/10 000 dilution (Cat No: 66868-1-Ig, Proteintech, China).

Techniques: Expressing

Journal: Frontiers in immunology

Article Title: Immunometabolic interference between cancer and COVID-19.

doi: 10.3389/fimmu.2023.1168455

Figure Lengend Snippet: FIGURE 9 Flow cytometry analysis of NAMPT and NAD-dependent enzymes in the different patient cohorts. Box-and-whisker plots representing FACS quantification of (A) NAMPT, (B) pSIRT1, (C) CD38, (D) CD39 and (E) PARP expression in the indicated immune cell populations (CD3+ T cells, CD19+

Article Snippet: Cells were incubated with the following antihuman antibodies for 20 min at 4°C: CD33 PerCP Cy5.5 (WM53) (0.6:100, Biolegend cat No. 303402), CD14 BV650 (M5E2) (1.25:100, Biolegend cat. No. 301835), CD16 BV711 (3G8) (0.3:100, Biolegend cat. No. 302044), HLA-DR BV421 (L243) (1.25:100, Biolegend Cat No. 307636), CD15 BV786 (HI98) (1.25:100, BD Biosciences cat. No. 563838), CD11c BUV661 (B-ly6) (0.6:100, BD Biosciences cat. No. 612968), CD123 BUV395 (7G3) (0.6:100, BD Biosciences cat. No. 564195), CD38 PE-Cy7 (HIT2) (1.25:100, Invitrogen cat. No. 25- 0389-42), CD39 PE (MZ18-23C8) (0.6:100, MACSMiltenyi Biotec Inc. cat. No.130-118-668), CD39 FITC (MZ18-23C8) (1.25:100, MACS Miltenyi Biotec Inc. cat. No. 130-125-113), CD3 BV650 (OKT3) (1.25:100, Biolegend cat. No.317324), CD4 BV570 (OKT4) (2.5:100, Frontiers in Immunology 03 Biolegend cat. No. 317445), CD8 BV786 (RPA-T8) (0.6:100, BD Biosciences cat. No. 557085), CD19 AF-700 (HIB19) (2.5:100, BD Biosciences cat. No. 561031), CD45RA APC-Cy7 (HI100) (1.25:100, BD Biosciences cat. No. 560674), CD45RO BUV395 (UCHL1) (1.25:100, BD Biosciences 564292), CD27 PE-Cy5 (O323) (1.25:100, Invitrogen cat. No. 15295964), CD16 BV605 (3G8) (0.6:100, Biolegend 302040), CD3 BUV496 (UCHT1) (1.25:100, BD Biosciences cat. No. 612940), CD66b PE-Cy7(G10F5) (0.6:100 Biolegend cat. No. 305116) and IgD FITC (IA6-2) (1.25:100, Invitrogen cat. No. 11-9868-42).

Techniques: Flow Cytometry, Whisker Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD39 , REA739 , 50 , 130-110-650 , PE , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Cellular neighborhood analysis of PD1 high/low T cells in the tumor margin and core. (A–D) Topology of PD1 high (left) and PD1 low (right) T cells and their cellular neighborhood within a 5 µm range. (A, B) represent tumor margin and (C, D) show tumor core areas. Cell types showing different distribution patterns around PD1 high and PD1 low T cells (mDCs, M1-like M, M2-like M, MDSCs, Fibroblasts, vessels, tumor cells) are highlighted by arrowheads. (E, F) Quantification of cells in a 5 µm range around of PD1 high/low T cells for tumor margin and tumor core, (E) represents immune cells and (F) stroma/tumor cells. (G) Violin plots for expression levels of eight immune-modulating markers (CD112, CD155, CD276, CD39, CD73, IDO, PD-L1, and VISTA) for the most important immune and tumor cells around PD1 high/low T cells in the tumor core area. Violin plots for tumor margin are shown in Supplementary Figure S5E . Depicted markers and annotated cell types as indicated by the color code. ROI sizes: Tumor margin (ROI15) and tumor core (ROI16): 975 x 769 µm.

Article Snippet: CD39 , REA739 , 50 , 130-110-650 , PE , Miltenyi Biotec.

Techniques: Expressing

Journal: Neurochemistry international

Article Title: Increased spinal adenosine after subacute cervical injury correlates with sustained upregulation of CD39 and CD73 in microglia

doi: 10.1016/j.neuint.2025.106030

Figure Lengend Snippet: CD39 mRNA was assessed in controls (uninjured) or 1-, 2- and 8-weeks post-C2 hemisection in isolated microglia and microglia-free homogenates at (A) and below (B) injury. (C) Flow cytometry analysis of CD39 protein. Example of CD39 in C1–C3 microglia in uninjured (upper left); 1-week post-injury (upper right); histogram of fluorescence intensity (bottom). Negative controls are cells stained with CD11b-PE antibody and secondary-Alexa405 antibody without CD39 antibody. (D) CD39 protein analysis for experimental groups. MFI, median fluorescent intensity. Controls (uninjured rats) were included for each time points 1-, 2-, 8-weeks after injury. Because there were no differences in control rats, data were pooled together into one ctrl bar. N = 4–8/group; **p < 0.01 vs ctrl, *p < 0.05 vs ctrl; +++p < 0.001 vs microglia.

Article Snippet: 2 , CD11b-PE (Miltenyi) , CD39 (Proteintech) , Goat anti-rabbit-Alexa 405 (Invitrogen).

Techniques: Isolation, Flow Cytometry, Fluorescence, Staining, Control

Journal: Neurochemistry international

Article Title: Increased spinal adenosine after subacute cervical injury correlates with sustained upregulation of CD39 and CD73 in microglia

doi: 10.1016/j.neuint.2025.106030

Figure Lengend Snippet: CD73 mRNA was assessed in controls (uninjured) or 1-, 2- and 8-weeks after C2 hemisection in isolated microglia and microglia-free homogenates at (A) and below (B) injury. (C) Flow cytometry analysis of CD73 protein. Example of CD73 in C1–C3 microglia in controls (top left) and 1-week post-injury (top right); with histogram of fluorescence intensity (bottom). Negative controls are cells stained with CD11b-PE antibody and secondary-Alexa405 antibody without CD73 antibody. (D) CD39 protein analysis for each experimental group. MFI, median fluorescent intensity. Controls (uninjured rats) were included for each time points 1-, 2-, 8-weeks after injury. Because there were no differences in control rats, data were pooled together into one ctrl bar N = 4–8/group C. CD39 mRNA expression in microglia in control and injured animals. N = 4/group; **p < 0.01 vs ctrl, *p < 0.05 vs ctrl; +++p < 0.001 vs microglia.

Article Snippet: 2 , CD11b-PE (Miltenyi) , CD39 (Proteintech) , Goat anti-rabbit-Alexa 405 (Invitrogen).

Techniques: Isolation, Flow Cytometry, Fluorescence, Staining, Control, Expressing

Journal: Neurochemistry international

Article Title: Increased spinal adenosine after subacute cervical injury correlates with sustained upregulation of CD39 and CD73 in microglia

doi: 10.1016/j.neuint.2025.106030

Figure Lengend Snippet: CD39 mRNA levels were correlated with the expression of other microglial genes after SCI at (A) and below (B) the injury site. Similar correlation analysis was performed for CD73 at (C) and below (D) injury. The strongest correlation was observed with P2Y12 in both regions.

Article Snippet: 2 , CD11b-PE (Miltenyi) , CD39 (Proteintech) , Goat anti-rabbit-Alexa 405 (Invitrogen).

Techniques: Expressing

Journal: Neurochemistry international

Article Title: Increased spinal adenosine after subacute cervical injury correlates with sustained upregulation of CD39 and CD73 in microglia

doi: 10.1016/j.neuint.2025.106030

Figure Lengend Snippet: (A) Adenosine was measured in ventral C3–C6 spinal segments in intact animals (ctrl) and 2- and 9-weeks post C2 hemisection (SCI). N = 6–8/group. **p < 0.01 vs ctrl. (B) There was no correlation between spinal adenosine and non-microglial CD39 and CD73 expression. However, adenosine levels strongly correlated with microglial CD39 and CD73 both on the protein (C) and mRNA levels (D) . Because spinal adenosine and CD73/CD39 expression were analyzed in different sets of animals we used mean (SE) for correlation analyses.

Article Snippet: 2 , CD11b-PE (Miltenyi) , CD39 (Proteintech) , Goat anti-rabbit-Alexa 405 (Invitrogen).

Techniques: Expressing

Journal: Neurochemistry international

Article Title: Increased spinal adenosine after subacute cervical injury correlates with sustained upregulation of CD39 and CD73 in microglia

doi: 10.1016/j.neuint.2025.106030

Figure Lengend Snippet: SCI-associated cell damage, tissue hypoxia, and inflammation increase extracellular ATP promoting inflammation and neuronal damage. Microglia upregulation of CD39 (converting ATP to ADP to AMP) and CD73 (converting AMP to adenosine (ADO)) increases adenosine levels determining the magnitude and mechanism of mAIH-induced phrenic motor plasticity. attenuating microglial inflammatory responses. Conversely, increasing levels of adenosine shift the serotonin/adenosine balance. Figure was created in Biorender .

Article Snippet: 2 , CD11b-PE (Miltenyi) , CD39 (Proteintech) , Goat anti-rabbit-Alexa 405 (Invitrogen).

Techniques:

Journal: Science Advances

Article Title: Dysregulation of ectonucleotidase-mediated extracellular adenosine during postmenopausal bone loss

doi: 10.1126/sciadv.aax1387

Figure Lengend Snippet: ( A to K ) Characterization of healthy (sham) and OVX animals 4 weeks after ovariectomy. (A) Immunofluorescence staining of CD73 (green) and (B) CD39 (red) in vertebrae of OVX animals. Nuclear staining (blue). Scale bars, 100 μm. Inset shows magnified image of bone surface. Yellow arrowheads indicate cells positive for CD73 or CD39 on bone surface. Scale bars, 50 μm. (C) Flow cytometric analysis of CD73 and CD39 membrane expression of hematopoietic cells from mouse BM cells 4 weeks after ovariectomy (OVX) and healthy controls. (D) Percentage and median fluorescence intensity of hematopoietic cells expressing CD73. (E) Percentage and median fluorescence intensity of hematopoietic cells expressing CD39. (F) Flow cytometric analysis of CD73 and CD39 membrane expression of nonhematopoietic cells from mouse BM cells 4 weeks after ovariectomy and healthy controls. (G) Percentage and median fluorescence intensity of nonhematopoietic cells expressing CD73. (H) Percentage and median fluorescence intensity of nonhematopoietic cells expressing CD39. (I) CD73 gene expression and (J) CD39 gene expression of cells from bone chips. (K) Extracellular adenosine concentration in BM plasma of sham and OVX animals. n = 5. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Sections were then incubated with primary antibody against CD39 (5 μg/ml; AF4398, R&D Systems), CD73 (5 μg/ml; AF4488, R&D Systems), and A2BR (1:200; MBS8207549, MyBioSource, San Diego, CA) in diluent solution (1%, w/v) and normal donkey serum (1%, v/v) in TBS overnight at 4°C.

Techniques: Immunofluorescence, Staining, Membrane, Expressing, Fluorescence, Gene Expression, Concentration Assay, Clinical Proteomics

Journal: Science Advances

Article Title: Dysregulation of ectonucleotidase-mediated extracellular adenosine during postmenopausal bone loss

doi: 10.1126/sciadv.aax1387

Figure Lengend Snippet: ( A ) Flow cytometric analyses and ( B ) quantification of CD73 and CD39 in osteoprogenitors in the absence or presence of E2 (100 nM) for 3 days. ( C to E ) Single (ESR1 or ESR2) or dual (ESR1 and ESR2) ER knockdown (KD) by siRNA in primary mouse osteoprogenitors and analyzed after 3 days. (C) Flow cytometric analyses of CD73 and CD39 after single knockdown (ESR1 or ESR2) and dual knockdown (ESR1 and ESR2). (D) Percentage of double-positive (CD73/CD39) cells in single knockdown and dual knockdown cells. (E) In vitro adenosine levels normalized by cell number in single knockdown and dual knockdown cells. Control (scrambled) siRNA concentration for single knockdown and dual knockdown are 5 and 10 nM, respectively. n = 5. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Sections were then incubated with primary antibody against CD39 (5 μg/ml; AF4398, R&D Systems), CD73 (5 μg/ml; AF4488, R&D Systems), and A2BR (1:200; MBS8207549, MyBioSource, San Diego, CA) in diluent solution (1%, w/v) and normal donkey serum (1%, v/v) in TBS overnight at 4°C.

Techniques: Knockdown, In Vitro, Control, Concentration Assay

Journal: Science Advances

Article Title: Dysregulation of ectonucleotidase-mediated extracellular adenosine during postmenopausal bone loss

doi: 10.1126/sciadv.aax1387

Figure Lengend Snippet: ( A ) Flow cytometric analyses and ( B ) quantification of CD73 and CD39 in primary mouse mononuclear cells undergoing osteoclast differentiation in the absence or presence of E2 (100 nM) for 3 days. ( C to E ) Single (ESR1 or ESR2) or dual (ESR1 and ESR2) ER knockdown by siRNA during macrophage differentiation for 3 days and subsequent osteoclast differentiation for 6 days. (C) Flow cytometric analyses of CD73 and CD39 after single knockdown (ESR1 or ESR2) and dual knockdown (ESR1 and ESR2). (D) Percentage of double-positive (CD73/CD39) cells in single knockdown and dual knockdown cells. (E) In vitro adenosine levels normalized by cell number in single knockdown and dual knockdown cells. Control (scrambled) siRNA concentration for single knockdown and dual knockdown are 5 and 10 nM, respectively. n = 4. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Sections were then incubated with primary antibody against CD39 (5 μg/ml; AF4398, R&D Systems), CD73 (5 μg/ml; AF4488, R&D Systems), and A2BR (1:200; MBS8207549, MyBioSource, San Diego, CA) in diluent solution (1%, w/v) and normal donkey serum (1%, v/v) in TBS overnight at 4°C.

Techniques: Knockdown, In Vitro, Control, Concentration Assay

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: Impact of high glucose on extracellular pyrophosphate metabolism. Aortic smooth muscle cells were cultured for one month in media containing either low (1 g/L) or high (4.5 g/L) glucose. A Measurement of extracellular pyrophosphate levels. B Extracellular pyrophosphate-to-ATP ratio. C , D Analysis of the gene expression of key enzymes involved in extracellular pyrophosphate metabolism, including eNTPD1, eNPP1, and TNAP, from isolated total RNA. (E) Immunoblot analysis of proteins associated with extracellular pyrophosphate metabolism. F , G Quantification of protein levels via ELISA, highlighting significant differences. The data are shown as the mean ± SEM, with data derived from 4 independent experiments, each containing 4 replicate plates. Statistical significance was determined via Student’s t test, with asterisks denoting significance levels: * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Gene Expression, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: High glucose levels impair the pyrophosphate-to-phosphate ratio. Aortic smooth muscle cells were incubated for one month in medium containing 1 g/L or 4.5 g/L glucose. A Autoradiograph displaying representative products from the hydrolysis of ATP (1 µmol/L ATP, 10 µCi/mL [γ 32 Pi]ATP) incubated with or without recombinant eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) or eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) enzymes. Enzymatic hydrolysis generated radiolabeled 32 PPi (32-pyrophosphate) and 32 Pi (32-phosphate), which, alongside unreacted [γ 32 Pi]ATP, were separated by thin-layer chromatography (TLC), as detailed in the section. B Representative time course of ATP hydrolysis showing the products released over time. C Synthesis of the pyrophosphate 32 PPi via hydrolysis of [γ 32 Pi]ATP (10 µCi/mL; 1 µmol/L ATP) in the absence or presence of 100 µmol/L SBI245 (a specific TNAP inhibitor) or inorganic pyrophosphatase (PPase). D The pyrophosphate-to-phosphate ( 32 PPi/ 32 Pi) ratio was quantified following hydrolysis of [γ 32 Pi]ATP (10 µCi/mL; 1 µmol/L ATP) under various conditions: in the absence of inhibitors (Control), in the presence of an ectonucleoside triphosphate diphosphohydrolase (eNTPD) inhibitor (INH, 200 µmol/L), or with the recombinant enzymes eNPP1 and eNTPD1 (100 ng/mL). Experiments were conducted in media containing either physiological (1 g/L) or elevated (4.5 g/L) glucose concentrations. D) Synthesis of 32 PPi by hydrolysis of [γ 32 Pi]ATP (10 µCi/mL and 1 µmol/L ATP). E Hydrolysis of 32 PPi (10 µCi/mL and 5 µmol/L PPi). The results are shown as the mean ± SEM (4 independent experiments with 4 plates per experiment). Student’s t test ( E, F ) or one-way ANOVA with Tukey’s post hoc test ( C, D ) was used for statistical analysis. Asterisks indicate a statistically significant difference compared with the control group: * P < 0.05; *** P < 0.001. ### Indicates a value of P < 0.001 compared with the control group (1 g/L)

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Autoradiography, Recombinant, Generated, Thin Layer Chromatography, Control

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: STZ-treated rats exhibit impaired extracellular pyrophosphate metabolism in the aortic wall. A A representative time course of ATP hydrolysis was conducted using a 1 µmol/L ATP solution containing 10 µCi/mL [γ- 32 P]ATP as a radiotracer. The products of hydrolysis, 32 PPi (32-pyrophosphate), 32 Pi (32-phosphate), and [γ- 32 P]ATP-, were separated and quantified via thin layer chromatography, as outlined in the section. B The synthesis of pyrophosphate (PPi) was analyzed by hydrolyzing 1 µmol/L ATP containing 10 µCi/mL [γ- 32 P]ATP as a radiotracer. The reactions were carried out in the absence or presence of either a specific TNAP inhibitor (SBI-425) or inorganic pyrophosphatase (PPase). C The ratio of 32 PPi to 32 Pi generated by ATP hydrolysis was calculated to assess the efficiency and specificity of pyrophosphate synthesis. D The synthesis of 32 PPi was evaluated by hydrolyzing 1 µmol/L ATP containing 10 µCi/mL [γ- 32 P]ATP. E The release of 32 Pi was measured following the hydrolysis of 5 µmol/L pyrophosphate, which contained 10 µCi/mL 32 PPi as a radiotracer. F Quantification of protein levels via ELISA. G , H Total RNA was isolated from rat aortas to evaluate the expression levels of key enzymes involved in extracellular pyrophosphate metabolism, including eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), and tissue-nonspecific alkaline phosphatase (TNAP) (panel G . Additionally, the expression of calcification-related proteins, such as matrix Gla protein (MGP) and osteopontin (OPN), was assessed (panel H). The data are shown as the mean ± SEM and represent data from 12–16 independent aortas. Statistical analyses were performed via Student’s t test. Asterisks indicate a significant difference with *** P < 0.001

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Thin Layer Chromatography, Generated, Enzyme-linked Immunosorbent Assay, Isolation, Expressing

Journal: Cell Death & Disease

Article Title: P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: Role of the A2A/CD39/CD73 axis

doi: 10.1038/s41419-025-07897-2

Figure Lengend Snippet: A CT26 CRC cells were stained with PKH26GL (red) and quinacrine (green) fluorescent dyes. Images were acquired using confocal microscopy at time 0 and after 5 min following P2X7 activation with 300 µM BzATP and are extrapolated from a 30-min time course (see supplementary videos and ). B CT26 cells were pre-treated with P2X7 antagonist AZ10606120 (5 µM) for 10 min before application of BzATP. C Number of vesicles released in 30 min from CT26 cells in PBS vehicle (PBS S-VS), following stimulation with P2X7 agonist ATP (P2X7-VS) or 10 min pretreatment with P2X7 antagonist AZ10606120 followed by stimulation with 3 mM ATP (AZ-VS) ( n = 5). D Size of PBS S-VS, P2X7-VS, and AZ-VS ( n = 5). E Number of vesicles released in 30 min from CT26 cells in DMSO vehicle (DMSO S-VS), following stimulation with P2X7 agonist ATP (P2X7-VS) or 10 min pretreatment with P2X7 antagonist A740003 followed by stimulation with 3 mM ATP (A74-VS) ( n = 7). F Size of DMSO- S-VS, P2X7-VS, and A74-VS ( n = 7). G Western blot for GM130, Alix, P2X7, CD39, CD73, and A2A in CT26 cells, S-VS and P2X7-VS. Pericellular ATP was measured with the pmeLUC probe expressed on the cell surface of untreated CT26 cells or after 5 min of exposure to PBS vehicle, S-VS, and P2X7-VS ( n = 4). H Quantification of luminescence changes was expressed as a fold increase on time 0. I Representative images of photon emissions. Changes in ATP J concentration increase on time 0 in the supernatants of CT26 cells, untreated or treated with PBS vehicle, S-VS, or P2X7-VS, measured with a luciferin/luciferase assay ( n = 3). Changes in adenosine K concentration increase on time 0 in the supernatants of CT26 cells untreated or treated with PBS vehicle, S-VS, P2X7-VS, or P2X7-VS plus 5uM CD73 inhibitor AB680 ( n = 5). * p < 0.05, ** p < 0.001, *** p < 0,0001, **** p < 0.00001.

Article Snippet: Tissue slides from the mouse lungs and rat colons were analyzed for P2X7, CD39, CD73, and A2A expression using the following primary antibodies: P2X7 1:100 (P8232, Sigma-Aldrich), CD39 1:100 (NBP2-67230, Novus Biologicals, Minneapolis, Minnesota, USA), CD73 1:500 (MAB5795, R&D Systems, Minneapolis, Minnesota, USA), and A2A 1:100 (SC32261, Santa Cruz Biotechnology).

Techniques: Staining, Confocal Microscopy, Activation Assay, Western Blot, Concentration Assay, Luciferase

Journal: Cell Death & Disease

Article Title: P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: Role of the A2A/CD39/CD73 axis

doi: 10.1038/s41419-025-07897-2

Figure Lengend Snippet: The mRNA expression of P2X7A A , B , P2X7B C , D , CD39 E , F , CD73 G , H , and A2A I , J was evaluated in the cDNAs of 158 patients with CRC subdivided into stage I ( n = 24), stage II ( n = 50), stage III ( n = 52), and stage IV ( n = 32) which comprised 11 samples derived from metastases in organs other than the colon. K Spearman’s correlation coefficient among P2X7A, P2X7B, CD39, CD73 , and A2A was evaluated in CRC metastatic patients. L Spearman’s correlation coefficient was evaluated between P2X7 and A2A expression in colon adenocarcinoma samples obtained from the Cancer Genome Atlas database. * p < 0.05, ** p < 0.01, *** p < 0.001. **** p < 0.0001.

Article Snippet: Tissue slides from the mouse lungs and rat colons were analyzed for P2X7, CD39, CD73, and A2A expression using the following primary antibodies: P2X7 1:100 (P8232, Sigma-Aldrich), CD39 1:100 (NBP2-67230, Novus Biologicals, Minneapolis, Minnesota, USA), CD73 1:500 (MAB5795, R&D Systems, Minneapolis, Minnesota, USA), and A2A 1:100 (SC32261, Santa Cruz Biotechnology).

Techniques: Expressing, Derivative Assay

Journal: Cell Death & Disease

Article Title: P2X7 a new therapeutic target to block vesicle-dependent metastasis in colon carcinoma: Role of the A2A/CD39/CD73 axis

doi: 10.1038/s41419-025-07897-2

Figure Lengend Snippet: mRNA expression of A P2X7A , B P2X7B , C A2A , D CD39 , and E CD73 in CRC patients subdivided into APC WT and APC mutated groups ( n = 6). Percentage of cells positive for P2X7 F and A2A G in the colons of WT and PIRC rats and PIRC tumors ( n = 4). Representative images of immunohistochemical staining for P2X7 and A2A in the colon of WT 1-year rats H, K and in the normal colon I, L and the tumor mass J, M of 1-year PIRC rats. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Tissue slides from the mouse lungs and rat colons were analyzed for P2X7, CD39, CD73, and A2A expression using the following primary antibodies: P2X7 1:100 (P8232, Sigma-Aldrich), CD39 1:100 (NBP2-67230, Novus Biologicals, Minneapolis, Minnesota, USA), CD73 1:500 (MAB5795, R&D Systems, Minneapolis, Minnesota, USA), and A2A 1:100 (SC32261, Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Staining