Journal: Frontiers in Immunology

Article Title: Human gut microbiota-reactive DP8α regulatory T cells, signature and related emerging functions

doi: 10.3389/fimmu.2022.1026994

Figure Lengend Snippet: Treg clone selection and characterization. The gating strategy to sort F. prausnitzii -reactive DP8α and classical FoxP3 + Tregs used for clone production is described in the Methods’ section and shown in Figure S1 . (A-D) Clones were screened by ELISA for their IFNγ (A, B) and IL-10 (C , D) responses to F. prausnitzii or anti-CD3 stimulation (clone OKT3). (E , F) FoxP3 expression was assessed by intracellular staining. Representative histogram plots examples (left) and all data (right) are shown (G, H) . CD39 (G) and CD73 (H) expression by clones measured using surface immunostaining. (I , J) . CD3/CD28-stimulated VPD-stained CD4 + T cells were co-cultured with Treg clones (ratio 1:1) in the presence or not of CD39 and CD73 inhibitors, namely POM-1 (30μM) and AB-680 (100nM), respectively, as indicated. Proliferation was evaluated by VPD dilution. Representative histogram plots examples (I) and all tested conditions (i.e., 5 DP8α clones and 2 FoxP3 + clones each co-cultured with CD4 + cells from 3 distinct donors) (J) are shown. (K) CD39 and CD73 expression by gated CD3 + /CD4 + /CD8α LOW /CCR6 + /CXCR6 + DP8α and CD3 + /CD4 + /CD25 HIGH /CD127 LOW classical Tregs among ex vivo PBMCs is shown. RFI corresponds to the ratio: MFI obtained with the relevant antibody over MFI for the corresponding isotype control. Mann-Whitney tests were used for single comparisons, while one-way ANOVA followed by Dunn’s multiple comparison tests were performed for 1J, p<.05 was considered statistically significant.

Article Snippet: POM-1 and AB-680 (MedChemExpress) drugs, which inhibit CD39 and CD73, respectively, were used as indicated in the figure legends.

Techniques: Selection, Clone Assay, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Immunostaining, Cell Culture, Ex Vivo, Control, MANN-WHITNEY, Comparison

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: Characterization of the immune landscape of EGFR -mutant NSCLC identifies CD73/adenosine pathway as a potential therapeutic target

doi: 10.1016/j.jtho.2020.12.010

Figure Lengend Snippet: CD73/adenosine pathway was upregulated in EGFR-mutant tumors. (A-D) NT5E (CD73), CD38, ADORA1 and ADORA2A RNA expression levels were compared between EGFR-mutant and WT tumors in PROSPECT, ICON and TCGA. Fold-change was calculated comparing expression of EGFR-mutant tumors to WT, using the lower expressed as the denominator. A positive fold-change value indicates overexpression in EGFR-mutant tumors and a negative value indicates decreased expression in EGFR-mutant tumors. The univariant analysis was used for p-value. (E) Feature plots showing EPCAM+ cells from the 3 single-cell RNAseq samples (right panel: cells by sample; middle panel: cells by malignant versus normal; left panel, the expression levels of NT5E in each cell) (F). Porportion of NT5E expression cells in each sample. (G) phosphorylated EGFR, CD73 and CD38 protein levels were compared between EGFR-mutant and WT tumors in the ICON cohort, from the reverse-phase protein array (RPPA).

Article Snippet: Treatment studies: Once the tumors were confirmed by MRI mice were either treated with vehicle (n=6) or CD73 blocking antibody (n=4) (BioXcell, in vivo monoclonal anti-mouse CD73 antibody clone: TY/23) twice a week, 100ug/dose per mouse.

Techniques: Mutagenesis, RNA Expression, Expressing, Over Expression, Protein Array

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: Characterization of the immune landscape of EGFR -mutant NSCLC identifies CD73/adenosine pathway as a potential therapeutic target

doi: 10.1016/j.jtho.2020.12.010

Figure Lengend Snippet: CD73 blockade modulates T cell composition and function in EGFR-mutant non-small cell lung cancer cells. (A) CD73 cell surface expression on H1975 cells was assessed by flow cytometry with or without control and CD73 siRNAs. (B) Healthy donor PBMCs were incubated with conditioned median collected from H1975 cells after siRNA treatment. T regulatory cell population was assessed in each condition by flow cytometry using CD4+ and FoxP3+ as markers. (C) Healthy donor PBMCs were incubated with conditioned median collected from H1975 cells or H1975 after recombinant CD73 overexpression. T regulatory cell population was assessed by flow cytometry using CD4+ and FoxP3+ as markers. (D) Healthy donor PBMCs were co-cultured with H1975 cells with anti-CD73 and anti-PD-1 treatment, alone or in combination. Percent of tumor cell lysis was assessed using cytotoxicity assay. (E) Healthy donor PBMCs were co-cultured with H1975 cells with anti-CD73 and anti-PD-1 treatment, alone or in combination. The cell medium was collected and assessed for interferon release by ELISA INF-gamma assay. Student t-test was used, statistical significance: * indicates p value less than 0.05, ** indicates p value less than 0.005, *** indicates p value less than 0.0005, and **** indicates p value less than 0.0001.

Article Snippet: Treatment studies: Once the tumors were confirmed by MRI mice were either treated with vehicle (n=6) or CD73 blocking antibody (n=4) (BioXcell, in vivo monoclonal anti-mouse CD73 antibody clone: TY/23) twice a week, 100ug/dose per mouse.

Techniques: Mutagenesis, Expressing, Flow Cytometry, Incubation, Recombinant, Over Expression, Cell Culture, Lysis, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: Characterization of the immune landscape of EGFR -mutant NSCLC identifies CD73/adenosine pathway as a potential therapeutic target

doi: 10.1016/j.jtho.2020.12.010

Figure Lengend Snippet: Anti-CD73 therapy-induced tumor reduction in EGFR-mutant murine lung cancers. (A) NT5E (CD73) and ADORA1 RNA expression levels in EGFR-mutant tumors and normal lung. (B) adenosine and AMP metabolite levels in EGFR-mutant tumors and normal lung. (C) CD73 immunohistochemistry for EGFR-mutant tumors and adjacent normal lung. (D) The change of tumor sizes with 2 weeks of treatment was documented and compared between vehicle-treated and anti-CD73 treated groups.

Article Snippet: Treatment studies: Once the tumors were confirmed by MRI mice were either treated with vehicle (n=6) or CD73 blocking antibody (n=4) (BioXcell, in vivo monoclonal anti-mouse CD73 antibody clone: TY/23) twice a week, 100ug/dose per mouse.

Techniques: Mutagenesis, RNA Expression, Immunohistochemistry

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: The Characteristics of Antibodies

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques:

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: RT-qPCR Primers

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques:

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: EtOH‑fed mice had higher levels of CD73 than pair‑fed mice. ( A ) The process of alcohol-induced liver injury and inflammation. ( B ) Body weight loss, n=6. ( C ) Liver index increases, n=6. ( D ) Serum ALT and AST levels, n=5–6. ( E ) Hepatic TG and T-CHO levels, n=5–6. ( F ) Representative H&E staining of liver sections (100 μm, 20 μm). ( G ) Representative Oil Red O staining of liver sections (100 μm, 20 μm). ( H ) Representative BODIPY staining of liver sections (50 μm, 20 μm). ( I ) The expression of IL-6 was detected by IHC (50 μm, 20 μm). ( J ) The expression of IL-1β was detected by IHC (50 μm, 20 μm). ( K ) The mRNA levels of IL-6 and IL-1β in the liver. ( L ) The release of the inflammatory cytokines IL-6 and IL-1β from serum was measured by ELISA. ** P < 0.01, *** P < 0.001 compared with the pair-fed group.

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques: Staining, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: The expression of CD73 was increased in EtOH-primed RAW264.7 cells ( A ) The protein level of CD73 in the liver. ( B ) The mRNA level of CD73 in the liver. ( C ) The expression of CD73 was detected by IHC (50 μm, 20 μm). ( D ) Double immunofluorescence staining of CD73 (red) and F4/80 (green); representative views from the pair-fed group and EtOH-fed group were presented (20 μm). * P < 0.05, ** P < 0.01 compared with the pair-fed group.

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques: Expressing, Double Immunofluorescence Staining

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: The expression of CD73 was increased in EtOH-primed RAW264.7 cells. ( A ) Effect of different concentrations of EtOH on RAW264.7 cell viability by CCK-8 assay. ( B and C ) The protein level of CD73 in RAW264.7 cells. * P < 0.05, ** P < 0.01 compared with 0 mM. ( D and E ) The protein level of CD73 in RAW264.7 cells. * P < 0.05 compared with 0 h. ( F and G ) The protein level of CD73 in RAW264.7 cells. ( H ) The mRNA level of CD73 in RAW264.7 cells. ( I ) The release of the inflammatory cytokines IL-6 and IL-1β from RAW264.7 cells into serum was measured by ELISA. ( J ) The mRNA levels of IL-6 and IL-1β in the liver. ** P < 0.01, *** P < 0.001 compared with the control group.

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques: Expressing, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: CD73 inhibited the secretion of IL-6 and IL-1β in RAW264.7 cells induced by EtOH. ( A ) The protein and mRNA levels of CD73 in RAW264.7 cells. ** P < 0.01 compared with the control group. ## P < 0.01, ### P < 0.001 compared with the pEX3-NC+EtOH group. ( B ) The protein and mRNA levels of CD73 in RAW264.7 cells. ** P < 0.01 compared with control group. ## P < 0.01, ### P < 0.001 compared with the scrambled-siRNA+EtOH group. ( C ) The levels of proinflammatory cytokines (IL-6 and IL-1β) in culture supernatants were determined by ELISA, and the mRNA levels of IL-6 and IL-1β in RAW264.7 cells. * P < 0.05, *** P < 0.001 compared with the control group. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the pEX3-NC+EtOH group. ( D ) The levels of proinflammatory cytokines (IL-6 and IL-1β) in culture supernatants were determined by ELISA, and the mRNA levels of IL-6 and IL-1β in RAW264.7 cells. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control group. # P < 0.05, ## P < 0.01 compared with the scrambled-siRNA+EtOH group.

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: rAAV9‑CD73 protected against alcohol-induced liver injury and inflammation. ( A ) Small animal in-vivo imaging system. ( B ) Liver index, n=6. ( C ) Serum ALT and AST levels, n=5–6. ( D ) Hepatic TG and T-CHO levels, n=5–6. ( E ) Representative H&E staining of liver sections (50 μm). ( F ) Representative BODIPY staining of liver sections (50 μm). ( G ) The expression of CD73 was detected by IHC (50 μm). ( H ) The mRNA level of CD73 in the liver. ( I ) The mRNA levels of IL-6 and IL-1β in the liver. ( J ) The protein levels of CD73, IL-6 and IL-1β in the liver. ( K ) The protein levels of CD73, IL-6 and IL-1β in primary macrophages. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the pair-fed group. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the rAAV9-empty-EtOH-fed group.

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques: In Vivo Imaging, Staining, Expressing

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: CD73 inhibits the apoptosis of RAW264.7 cells. ( A ) The effect of increased CD73 on the apoptosis of EtOH-activated RAW264.7 cells was determined by flow cytometry. ** P < 0.01 compared with the pEX3-NC+EtOH group. ( B ) Expression of Bax, Bcl-2 and cleaved caspase-3 in RAW264.7 cells transfected with the pEX3-CD73. * P < 0.05, *** P < 0.001 compared with the control group. # P < 0.05, ### P < 0.001 compared with the pEX3-NC+EtOH group. ( C ) The effect of decreased CD73 on the apoptosis of EtOH-activated RAW264.7 cells was determined by flow cytometry. * P < 0.05 compared with the scrambled-siRNA+EtOH group. ( D ) Expression of Bax, Bcl-2 and cleaved caspase-3 in RAW264.7 cells transfected with CD73-siRNA. ** P < 0.01 compared with the control group. # P < 0.05, ## P < 0.01 compared with the Scrambled-siRNA+EtOH group.

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques: Flow Cytometry, Expressing, Transfection, Control

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: CD73 promotes the proliferation of RAW264.7 cells. ( A ) The effect of increased CD73 on the proliferation of EtOH-activated RAW264.7 cells was determined by flow cytometry. ( B ) Expression of c-Myc and CyclinD1 in RAW264.7 cells transfected with pEX3-CD73. ** P < 0.01 compared with control group. ## P < 0.01 compared with the pEX3-NC+EtOH group. ( C ) The effect of decreased CD73 on the proliferation of EtOH-activated RAW264.7 cells was determined by flow cytometry. ( D ) Expression of c-Myc and CyclinD1 in RAW264.7 cells transfected with CD73-siRNA. * P < 0.05 compared with control group. # P < 0.05 compared with the scrambled-siRNA+EtOH group.

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques: Flow Cytometry, Expressing, Transfection, Control

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: Overexpression of CD73 blocked the TLR4/MyD88/NF-κB signaling pathway. ( A and E ) The protein levels of TLR4, NF-κB, p-NF-κB and MyD88 in the liver. * P < 0.05, ** P < 0.01 compared with the pair-fed group. ( B, C, D, F, G and H ) The protein levels of TLR4, NF-κB, p-NF-κB and MyD88 in RAW264.7 cells. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control group. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with pEX3-NC+EtOH/ scrambled-siRNA+EtOH group.

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques: Over Expression, Control

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: CD73 regulates the inflammatory response and apoptosis in EtOH treated RAW 264.7 cells through TLR4/MyD88/NF-κB. ( A ) Effect of different concentrations of TAK242 on RAW264.7 cell viability by CCK-8 assay. * P < 0.05, ** P < 0.01 compared with 0 ng/mL. ( B ) The mRNA level of TLR4 in RAW264.7 cells. ** P < 0.01 compared with 0 ng/mL. ( C ) The protein levels of IL-6 and IL-1β in RAW264.7 cells. ( D ) Expression of Bax, Bcl-2 and cleaved caspase-3 in RAW264.7 cells. ( E ) The apoptosis of RAW264.7 cells was determined by flow cytometry. ( F ) Expression of c-Myc and CyclinD1 in RAW264.7 cells. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the pEX3-NC+EtOH group. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the pEX3-CD73+TAK242+EtOH group. ( G ) The proliferation of RAW264.7 cells was determined by flow cytometry.

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques: CCK-8 Assay, Expressing, Flow Cytometry

Journal: Journal of Inflammation Research

Article Title: CD73 Attenuates Alcohol-Induced Liver Injury and Inflammation via Blocking TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.2147/JIR.S341680

Figure Lengend Snippet: CD73 inhibited the release of inflammatory cytokines and RAW264.7 cell apoptosis through downregulation of the TLR4/MyD88/NF-κB signaling pathway.

Article Snippet: The sections were treated with 0.3% hydrogen peroxide for 15 min, blocked with 2% bovine serum albumin, and then blocked with anti-L-1β (1:500, Bioss, China), IL-6 (1:500, Bioss, China) and CD73 (1:500, Proteintech, USA) primary antibodies were incubated overnight.

Techniques:

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: CD73 was overexpressed in the tumour cells derived sEVs of HNSCC patients. (a) Schematic of the progress of extracting sEVs from Conditional Reprogramming (CR) cells. (b) The histological and morphological characteristics of CR coculture of keratinocytes and feeder cells from HNSCC and adjacent normal tissues. (c) Electron microscopy images of sEVs from HNSCC. Scale bar, 100 and 200 nm. (d) Nanoparticle tracking analysis (NTA) of sEVs from HNSCC. (e) Immunoblotting for sEVs biomarkers. (f) Quantitative proteome results of cells released sEVs from HNSCC or adjacent normal tissues, shown by venn diagram. (g) The mass spectrum identified CD73 in sEVs derived from HNSCC cells. (h) Representative western blotting for CD73 in sEVs from six pairs of HNSCC samples

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Derivative Assay, Electron Microscopy, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: CD73 in sEVs was closely contributed to tumour associated macrophages and HNSCC malignant progress. (a) IHC analysis of CD73 expression levels in tissue microarrays, containing 10 normal tissues and 92 HNSCC tissues. Representative immunohistochemistry images of normal tissue, weak positive, modest positive, and strong positive CD73 staining were shown. Scale bar: 200 μm, 40 μm. (b) Statistical analysis about lymph node metastasis (LN metastasis), tumour stage, pathologic stage and overall survival rate with CD73 stain intensity in HNSCC tissues. (c) The correlation of NT5E expression with immune infiltration level in HNSCC investigated in TCGA database based on six deconvolution algorithms. (d) Immunofluorescence staining of CD73 distribution (green) and different resident immune‐associated cell types (red), including macrophages (CD68 + ), CD4 + T cells, CD8 + T cells, Tregs (Foxp3 + ) and CAFs(α‐SMA + ) in tissues from HNSCC patients. Scale bar: 40 μm. (e) The percentage of costaining of CD73 with macrophages, CD4 + T cells, CD8 + T cells, Tregs and CAFs in HNSCC patient tumour samples. (f) NT5E expression ( NT5E high , NT5E low ) as a marker for prediction of overall survival rate in TCGA HNSCC cohort. Data were classified into low macrophage/low M2 macrophage signature (Mac low /M2 low ) and high macrophage/high M2 macrophage signature (Mac high /M2 high ). Log‐rank Mantel‐Cox test was used to assess significance. (g) The association between sensitivity of anti‐PD‐L1 treatment and NT5E expression were studied through the public data of IMvigor210CoreBiologies, lower NT5E group exhibited increased sensitivity to PD‐L1 blockade than higher NT5E group. (h) The percentage of SCC7 Nt5e OE‐GFP ‐derived sEVs CD73‐GFP signal that distributed on macrophages (F4/80 + ), CD4 + T cells, CD8 + T cells, Tregs (Foxp3 + ) in SCC7 tumour‐bearing C3H mice. (i) The SCC7 Nt5e OE‐GFP ‐derived sEVs CD73‐GFP were injected into tumours on C3H mice. After 24 h, fluorescence visualization identified the coexpression of CD73‐GFP in sEVs (green) with immunocytes(red) in tumours. Scale bar: 20 μm. (j) Schematic of sEVs injection through foot pad and its draining lymph node (DLNs). (k) Fluorescence microscopy showed sEVs CD73‐GFP (green) in whole DLNs imaging after 30 or 60 min of sEVs CD73‐GFP (10 μg) injection. Scale bar: 200 μm, 50 μm. (l) Flow cytometry analysis for subpopulation in CD73‐GFP + cells of DLNs (sEVs CD73‐GFP : 25 μg, 24 h). (m) The sEVs SCC7 (25 μg) were injected into foot pads. After 24 h, flow cytometry analyzed the expression of CD73 + or PD‐1 + immunocytes in DLNs. (n) Flow cytometry analysis for percentage of macrophages in DLNs after injecting sEVs (25 μg) derived from SCC7 cells (sEVs SCC7 ), mBMSC cells (sEVs mBMSC ) or mBMSC‐ Nt5e OE cells (sEVs mBMSC‐ Nt5e OE ) every day. DLNs were harvested at different time point. Lymph nodes from untreated mice were used as normal control. CD4 + T: CD4 + T cells, CD8 + T: CD8 + T cells, Mac: Macrophages. Data were analysed by Mann‐Whitney test. (ns, no significant difference, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Expressing, Immunohistochemistry, Staining, Immunofluorescence, Marker, Derivative Assay, Injection, Fluorescence, Microscopy, Imaging, Flow Cytometry, Control, MANN-WHITNEY

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: Relationship between CD73 level and clinicopathologic features in HNSCC tumour tissues ( n = 92)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Expressing, Significance Assay

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: The effect of CD73 in sEVs derived from HNSCC cells on the function of macrophages. (a) Macrophages were cocultured with DMEM or two HNSCC lines (SCC25, HN6) with or without NT5E/RAB27AKO for 24 h. HNSCC NT5E OE , referred to overexpression of NT5E performed on HNSCC NT5E KO cells. (b) Flow cytometry analysis for percentage of CD73 + macrophages and M2 macrophages (CD163 + CD206 + ) in coculture system. (c–i) The sEVs (50 μg) were cocultured with macrophages (1 × 10 6 ) for 24 h. The sEVs HNSCC derived from two HNSCC lines: SCC25 and HN6. The sEVs hBMSC‐ NT5E OE derived from hBMSC cells with NT5E overexpression. Latrunculin A (Lat A, 30 μM) was used as the inhibitor of sEVs uptaken. (c) Compromised phagocytosis of M2 macrophages treated with or without sEVs or Lat A. The percentage of pHrodo dyes of M2 macrophages was analyzed by flow cytometry. (d) The percentage of CD73 + macrophages and M2 macrophages after macrophages cocultured with sEVs. (e, f) Concentration of IL‐6, IL‐10, TNF‐α, and TGF‐β1 levels in macrophages conditional medium after sEVs education by ELISA. (e)The sEVs were derived from HNSCC cells or HNSCC NT5E KO cells. (f) The sEVs were derived from HNSCC, hBMSC or hBMSC NT5E OE cells with or without Lat A (30 μM). (g) Macrophages were cocultured with anti‐CD73‐FITC labelled sEVs from HNSCC cell lines control or RAB27A KO, CD73‐GFP labelled sEVs from hBMSC treated with or without Lat A were cocultured with macrophages for 1 h, and Laser Scanning Confocal Microscopy was used to analyze the internalization of HNSCC‐derived sEVs into macrophages (Scale bar = 25 μm). (h) Flow cytometry analysis for differential expression of immune check point (PD‐1, PD‐L1, LAG3, CTLA‐4, VISTA) comparing M0 and M2 macrophages which were educated with sEVs from HNSCC cells (h) or hBMSC with or without Lat A (i). Data were analysed by Mann‐Whitney test (ns, no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Derivative Assay, Over Expression, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Confocal Microscopy, Quantitative Proteomics, MANN-WHITNEY

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: Absence of CD73 in sEVs rescues immune suppression and restrains tumour growth in vivo. (a) Schematic of subcutaneous tumorigenesis in vivo experiment, followed with intratumoral injection of sEVs which were collected from SCC7, SCC7‐ Nt5e KO or SCC7‐ Nt5e OE cells grown in vitro. (b) The exhibition of isolated tumours. (c and d) The tumour weight and the time course of tumour growth in grams for 15 days postinjection with SCC7 or SCC7 Rab27a KO cells with or without CD73 in sEVs. Lat A was used as inhibitor of sEVs uptaken. (e) Flow cytometry analysis for infiltration of macrophages and percentage of CD73 + /PD‐1 + macrophages in tumours. (f). Flow cytometry analysis for infiltration of CD8 + T cells and percentage of CD73 + /PD‐1 + CD8 + T cells in tumours. (g) Flow cytometry analysis for infiltration of Tregs and percentage of CD73 + /PD‐1 + Tregs in tumours. (h) Schematic of subcutaneous tumorigenesis followed with intratumoral injection of engineered sEVs from mBMSC or mBMSC Nt5e OE . (i) The exhibition of dissected tumours. (j and k) The tumour weight and tumour growth curve of SCC7 control or SCC7 Rab27a KO with indicated treatment. (l and m) Flow cytometry analysis for infiltration of macrophages and percentage of CD73 + macrophages in tumours. (n) Flow cytometry analysis for infiltration of CD8 + T cells and Tregs in tumours. Data were analysed by Mann‐Whitney test (ns, no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: In Vivo, Injection, In Vitro, Isolation, Flow Cytometry, Control, MANN-WHITNEY

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: CD73 in sEVs regulates the immune functions of TAMs through NF‐κB pathway. (a) Venn diagram showing the differential expressed gene (DEG) of M2 macrophages depending on the regulation by sEVs CD73 or sEVs NT5E KO , the numbers of shared and exclusive genes were exhibited. (b) Bubble plot showing the predicted transcription factors of shared DEGs from AC. The shared DEGs and the signalling pathways which they belonged to were shown in circos gram, the number of genes was represented by node size. (d and e) GSEA analysis of up‐stream events of NF‐κB regulation. IκB phosphorylation and NIK signaling activity was measure after sEVs CD73 (up regulated) or sEVs NT5E KO (down regulated) incubation. (f) M2 macrophages were treated by HNSCC cell lines‐derived sEVs with or without CD73 for 3 h, with or without pretreatment of 100 μM PDTC for 1h. IF was applied for assessing the translocation of p65 in M2 macrophages (Scale bar = 40 μm). (g) Western blot was performed to validate the status of IκBα degradation, IκBα phosphorylation, p65 and phosphorylated p65 in M2 macrophages following treatment with sEVs or sEVs NT5E KO from HNSCC cell lines. (h) The heatmap showed the mRNA levels of IL6 , IL10 , TNFA , TGFB1 , CD274 , CD279 , and LAG3 in M2 macrophages which were stimulated by HNSCC derived sEVs, the expressions were partially downregulated when sEVs NT5E KO were added, and further inhibited when pretreated with PDTC

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Phospho-proteomics, Activity Assay, Incubation, Derivative Assay, Translocation Assay, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: The sEVs CD73 predicts HNSCC metastasis and targeting sEVs CD73 abolishes immunotherapy resistance. (a) Schematic of the circulating sEVs from HNSCC patients processing. (b) Percentage of CD73 + macrophages in PBMC. (c) The concentration of CD73 in serum sEVs (log2 TPM) detected by ELISA from HNSCC patients. (d) Level of CD73 (log2 TPM) on serum sEVs predicts higher risk of lymph node metastasis and larger tumour size. (e) CD73 indicated as a potential candidate to predict anti‐PD‐1 therapy response for HNSCC patients compared to other existed biomarkers, analysis by TIDE framework ( http://tide.dfci.harvard.edu ), TIDE, tumour immune dysfunction and exclusion. (f) The exhibition of isolated tumours 15 days after injection of SCC7 or SCC7 Rab27a KO cells with or without CD73 absent in sEVs combined with anti‐PD‐1 therapy. (g and h) The tumour weight and tumour growth curve of SCC7 control or SCC7 Rab27a KO with indicated treatment combined with anti‐PD‐1 therapy. (j) Flow cytometry analysis for infiltration of macrophages and percentage of CD73 + macrophages in tumours. (k) Flow cytometry analysis for infiltration of CD8 + T cells and Tregs in tumours. Data were analyzed by Mann–Whitney test (ns, no significant difference, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation, Injection, Control, Flow Cytometry, MANN-WHITNEY

Journal: Journal of Extracellular Vesicles

Article Title: CD73 in small extracellular vesicles derived from HNSCC defines tumour‐associated immunosuppression mediated by macrophages in the microenvironment

doi: 10.1002/jev2.12218

Figure Lengend Snippet: Relationship between sEVs CD73 in serum and clinicopathologic features ( n = 54)

Article Snippet: A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.

Techniques: Expressing, Significance Assay

Journal: bioRxiv

Article Title: The extracellular ATP/P2X7R signaling axis drives early neuroinflammation and neuronal hyperexcitability in an Alzheimer’s disease mouse model

doi: 10.1101/2025.11.14.688405

Figure Lengend Snippet: a , Representative immunoblot of NLRP3 protein, and relative quantification, in cortical lysates from 6-month-old (6 mo) WT (n = 8), AD (n = 8), and AD-P2X7RKO (n = 4) mice. Data are presented as mean ± s.e.m., normalized first to GAPDH and then to WT. b–d, Levels of IL-1β (b), TNF-α (c), and IL-6 (d) cytokines in SSCx from 6-month-old WT (n = 3), AD (n = 3), and AD-P2X7RKO (n = 4) mice, measured by ELISA. e–g , Quantitative PCR analysis of P2rx7/ P2X7R (e), Entpd1 /CD39 (f), and Nt5e /CD73 (g) expression in brain cortex from WT, AD, and AD-P2X7RKO mice at 2 mo (WT, n = 6; AD, n = 6; AD-P2X7RKO, n = 6) and 6 mo (WT, n = 6; AD, n = 6; AD-P2X7RKO, n = 3 for P2rx7, n = 4 for Entpd1 ). Data are presented as mean ± s.e.m.; statistical analysis was performed using Kruskal–Wallis test followed by Dunn’s post hoc test. For Nt5e (g), comparisons between WT and AD were performed using unpaired two-tailed t-tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: A NanoDropTM ND-1000 UV-Vis spectrophotometer (Isogen Life Science B.V., Utrecht, Netherlands) was used to quantify RNA. cDNA was synthesized from 1 μg of RNA using the RT First Strand Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. qPCR analysis was performed with the Gene expression master mix (Thermo Fisher Scientific) using probes for mouse p2rx7 (assay no. Mm00440578, Applied Biosystems, Foster City, CA, USA), Entpd1 (assay no. Mm00515447, Applied Biosystems), Nt5e (assay no. Mm 00501910, Applied Biosystems) genes.

Techniques: Western Blot, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Combining gemcitabine and MSC delivering soluble TRAIL to target pancreatic adenocarcinoma and its stroma

doi: 10.1016/j.xcrm.2024.101685

Figure Lengend Snippet:

Article Snippet: PE mouse anti-human CD73 , Miltenyi Biotec , REA804.

Techniques: Control, Virus, Variant Assay, Luciferase, Recombinant, Saline, Infection, Blocking Assay, Proliferation Assay, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Gene Expression, Software

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: A. Activity determination of recombinant human 5’-Nucleotidase/CD73 using increasing concentrations of enzyme and 10µM AMP substrate. The reaction was carried out at 23°C for 30min using AMP-Glo Assay System as described in Materials and Methods section. Activity of CD73 is monitored by how much AMP has been consumed in (A), i.e., RLU corresponds to the amount of AMP remaining and thus the activity of the enzyme is reciprocally correlated with RLU (see Schematic 1A); (B) Net RLU after subtracting the control (no-enzyme) from the RLU values at each point of enzyme concentration. The experiment was done in triplicates; results shown are mean ± SD. SD, standard deviation

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Recombinant, Glo Assay, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Time course study using recombinant human 5’-Nucleotidase CD73 protein titration and 25µM AMP for 5,10, and 20 minutes reaction time at 23°C. Activity was determined using AMP-Glo assay. Data are shown as net RLU after subtracting values for no enzyme control from the RLUs values at each point of enzyme concentration. EC 50 represents the amount of enzyme required for 50% maximal activity. Each point represents the average of triplicates; error bars represent SD

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Recombinant, Titration, Activity Assay, Glo Assay, Concentration Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Determination of Km value for AMP using 0.01ng recombinant human 5’-Nucleotidase/CD73 protein per reaction and varying AMP concentrations for 5min reaction at 23°C followed by AMP-Glo assay protocol. Data shown as net RLU vs. AMP concentration. The experiment was done in triplicates; results shown are mean ± SD.

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Recombinant, Glo Assay, Concentration Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Reactions contained 0.1 ng of recombinant human 5’-Nucleotidase/CD73 protein and either 5µM or 10µM AMP and different concentrations of inhibitor (AMP-CP). Reactions were carried out for 5min at 23°C and activity was monitored using AMP Glo assay. Experiments were done in triplicates, and results are shown as mean ± SD.

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Recombinant, Activity Assay, Glo Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Purified CD73 (0.1 ng/Rx) and cN-II (0.6 mU/Rx) were tested for inhibition by various concentrations of AMP-PC using 10µM AMP substrate at 23°C for 5 min (CD73 and 30 min (cN-II). Activities were determined following AMP-Glo protocol. The inhibitor inhibited CD73 activity with IC50 value of 3×10 -7 M with minimal or no inhibition of cN-II. Experiments were done in triplicates; results shown are mean ± SD

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Purification, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Five cell lines were evaluated for their CD73 enzyme activity in intact cells using AMP Glo protocol for cells as described in the method section. (A) Time course study of enzyme activity of five cell lines with 10 μM AMP using AMP-Glo for cell bound CD73. T-47D (circle), MDA-MB-231(square), SK-MEL2 (triangle), A375 (reverse triangle), and SK-OV3 (diamond). (B) Determination of abundance of CD73 in membranes of five cell lines using western blotting. Cell lysates (10µg) from each cell line and pure CD73 (0.2µg) as positive control (lane 7) were run on gels and immunoblotted using primary antibodies (anti- NT5E/CD73, Cell Signaling), incubated overnight at 4°C followed by HRP-ECL as described in the method section

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Western Blot, Positive Control, Incubation

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Membrane associated CD73 bound MDA-MB-231 activity was determined in presence (open circle) and absence (closed circle) of 50µM AMP-PC. Reactions contained 25k MDA-MB-231 cell per well and incubated at 37°C with 10µM AMP in final 250µl per well. Aliquots of 25µl per sample were withdrawn at each time point and activity was determined following AMP-Glo assay. Each point represents the average of triplicates; the error bars represent the SD.

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Membrane, Activity Assay, Incubation, Glo Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Three compounds with inhibitory activity against CD73 (AMP-PC, solid circle) and against CD39 (ARL 67156 and POM 1, square and triangle, respectively) were tested using purified CD73 and MDA-MB-231 bound CD73. (A) Purified CD73, 0.1ng per reaction with 1µM AMP for 5 min reaction at 23°C, and (B) cell-based MDA-MB-231, 25K cells per well with 5µM AMP for 90min at 37°C. Each point represents the average of triplicates; the error bars represent the SD

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Purification

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Commercially available antibodies against CD73 were tested using cell-based membrane-associated CD73 from MDA-MB-231 following AMP-Glo cell-based assay. (A) Using 9 different commercially available antibodies against CD73 with an antibody against CD39 as a negative control. Antibodies were applied at 10µg/ml per well and incubated with MDA-MB-231 cells for 1, 3, 6, and 16 hrs. Activities are expressed as percentage of control (cells without antibodies). (B) Effect of antibodies concentration of the most effective antibodies (Ab4 and Ab5) and the control CD39 selective antibodies (Ab10) on the activity of membrane bound CD-73. Activities are expressed as percentage of control (cells without antibodies). All antibodies were tested using 25K cells per well cells and incubated at 37°C. Activities were determined in the presence of 10µM AMP substrate for 30min at 37°C. Each point represents the average of triplicates; the error bars represent the SD

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Membrane, Cell Based Assay, Negative Control, Incubation, Concentration Assay, Activity Assay

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Inhibitors of CD39 and CD73 were incubated with purified CD39 (A, B) and Farage associated enzyme (C, D) and assayed for CD39 activity using ATP (A, C) or ADP (B, D) as substrate. Results show percentage inhibition of CD39 activity using ARL 67156 (circle), POM1 (solid square), and AMP-CP (triangle). Purified CD39 (0.1ng) and 10µM ATP or ADP in each reaction for 30min at 37°C; for cell-based Farage cells (200K cells) were incubated with 10µM ATP or ADP in each reaction for 20min at 37°C. Experiments were carried out in triplicates; results shown are mean ± SD

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Incubation, Purification, Activity Assay, Inhibition

Journal: bioRxiv

Article Title: Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

doi: 10.1101/698373

Figure Lengend Snippet: Testing the specificity of CD39 inhibitor POM1 (A) and CD73 inhibitor AMP-CP (B) on the activity of different kinases, ATPase, as well as CD39 and CD73. Enzyme tested are protein serine/threonine/tyrosine, lipid, sugar, and inorganic kinases, and K/Na ATPase. Results show that the majority of enzymes are inhibited by POM1 (A), but the more selective CD73 inhibitor AMP-PC) was highly selective for CD73 with minimal inhibition for PKA and no inhibition of the other enzymes. PKA, protein kinase A (solid circle), PKC□ (solid square), Src, protein tyrosine Src kinase (solid triangle), Acka, acetate kinase from Escherichia coli (solid reverse triangle), HK, hexokinase from Saccharomyces cerevisiae (solid circle), PI3□ Kinase, p110□/p85□ (square), K/K ATPase, Adenosine 5’-Triphosphatase from porcine cerebral cortex (circle), CD39 (ATP substrate, triangle), and CD39 (ADP substrate, reverse triangle); for CD73 (triangle) in penal B. Each point represents average of a typical experiment done in triplicates; results shown are mean ± SD.

Article Snippet: Active CD73 enzyme (Recombinant Human 5’-Nucleotidase/CD73 Protein, CF) and active CD39 (His-Tag Human) were from R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Inhibition