Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: A Flow cytometry results of CD73-positive ADSCs. B Flow cytometry results of CD73-negative ADSCs. C In vitro, immunofluorescence staining of ADSCs in each group. Red indicates CD73, green indicates VEGF, blue indicates DAPI, scale bar = 20 µm. D Quantitative analysis of immunofluorescence staining results among different groups ( n = 3). E Western blot results of CD73 and VEGF proteins after transfection or APCP treatment in each group. F Quantitative analysis of Western blot results. G CCK-8 assay results among different groups of ADSCs in conditioned medium ( n = 3). H Colony formation assay results among RBSMC in conditioned medium ( n = 3). I Cell migration assay results and quantitative analysis among different groups of ADSCs in conditioned medium ( n = 3). J Wound healing assay results among RBSMC in conditioned medium ( n = 3). Data presented as ±SD. * and # indicates P < 0.05, ** and ## indicates P < 0.01, and *** and ### indicates P < 0.001. Full-length blots/gels are presented in Supplementary Fig. .

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Flow Cytometry, In Vitro, Immunofluorescence, Staining, Western Blot, Transfection, CCK-8 Assay, Colony Assay, Cell Migration Assay, Wound Healing Assay

Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: A Western blot results of CD73 and VEGF in the bladder tissues of NB + CD73⁺ / ⁺ group on days 0, 7, 14, 21, and 28 after treatment. B Quantitative analysis of the Western blot results for CD73 and VEGF ( n = 3). C The results of the mean pressure of the voiding contractions and mean intermicturition interval in each group of rats ( n = 5). D Representative images of cystometrography results for each group of rats ( n = 5). E Masson staining results of bladder tissues in each group of rats. scale bar = 50 µm. F Immunofluorescence staining results for CD73 and VEGF in the bladder tissues of each group. Red indicates CD73, green indicates VEGF, and blue indicates DAPI. scale bar = 20 µm. G Quantitative analysis of smooth muscle content in the bladder (yellow square) of each group ( n = 3). H Quantitative analysis of immunofluorescence staining for CD73 and VEGF ( n = 3). Data presented as ±SD. * and # indicates P < 0.05, ** and ## indicates P < 0.01, and *** and ### indicates P < 0.001. Full-length blots/gels are presented in Supplementary Fig. .

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Western Blot, Staining, Immunofluorescence

Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: A Immunofluorescence staining results of ADSCs in the bladder tissues of each group of rats. Red represents ADSCs, green represents βIII-tubulin, and blue represents DAPI. Scale bar = 20 µm. B Immunohistochemical results of CXCR4 in the bladder tissues of each group. The green arrows indicate regions of high CXCR4 expression. Scale bar = 50 µm. C Immunofluorescence staining results of CD73 and SDF-1 in the bladder tissues of each group. Red represents SDF-1, green represents CD73, and blue represents DAPI. Scale bar = 20 µm. D Quantitative results of ADSCs in the bladder tissues of each group ( n = 5). E Quantitative results of SDF-1 expression in the bladder tissues of each group ( n = 3). F In vitro, western blot results of SDF-1 in each group under conditioned medium. G Quantitative results of SDF-1 expression of each group ( n = 3). H Cell migration assay results of CD73⁺ ADSCs after SDF-1 gene knockdown. I Quantitative results of cell migration assays for ADSCs in each group ( n = 3). Data presented as ±SD. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Full-length blots/gels are presented in Supplementary Fig. . Full-section IHC are presented in Supplementary Fig. .

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Expressing, In Vitro, Western Blot, Cell Migration Assay, Knockdown, Migration

Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: CD73 activation upregulates VEGF expression, further stimulating the PI3K/AKT/mTOR pathway to enhance cell proliferation. Simultaneously, it inhibits NFκB phosphorylation, suppressing the NFκB/NLRP3/caspase-1 axis, thereby preventing apoptosis and reducing IL-1β and IL-6 levels. Moreover, activated CD73 increases SDF-1 expression, which interacts with its receptor CXCR4 to direct cell migration to damaged bladder tissue.

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Migration

Journal: Frontiers in Pharmacology

Article Title: Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors

doi: 10.3389/fphar.2020.01294

Figure Lengend Snippet: Consecutive hydrolysis of ATP to adenosine by cleaving the terminal phosphate group and releasing inorganic phosphate (P i ), catalyzed by the enzymes CD39 and CD73.

Article Snippet: The cDNAs of the human enzymes NPP1, NPP3, NPP5, CD38 and CD73 (Genbank accession no. NM_006258, NM_005021, NM_021572, NM_ 001775, and NM_002526, respectively) were obtained from Origene (Rockville, USA).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors

doi: 10.3389/fphar.2020.01294

Figure Lengend Snippet: Inhibition of selected ecto-nucleotidases by ARL67156 (I) and analogs 31 and 33 ^a .

Article Snippet: The cDNAs of the human enzymes NPP1, NPP3, NPP5, CD38 and CD73 (Genbank accession no. NM_006258, NM_005021, NM_021572, NM_ 001775, and NM_002526, respectively) were obtained from Origene (Rockville, USA).

Techniques: Inhibition

Journal: Frontiers in Pharmacology

Article Title: Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors

doi: 10.3389/fphar.2020.01294

Figure Lengend Snippet: Comparison of (A) the putative substrate binding site of human CD39 (pale cyan) and (B) the substrate binding site of human CD73 (gray). Important amino acids are shown; positively and negatively charged amino acids are highlighted by blue and green boxes, respectively. Oxygen atoms are colored in red, nitrogen atoms in blue, sulfur atoms in yellow, calcium atom in green, and zinc atoms in dark gray.

Article Snippet: The cDNAs of the human enzymes NPP1, NPP3, NPP5, CD38 and CD73 (Genbank accession no. NM_006258, NM_005021, NM_021572, NM_ 001775, and NM_002526, respectively) were obtained from Origene (Rockville, USA).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors

doi: 10.3389/fphar.2020.01294

Figure Lengend Snippet: Docked poses of ARL67156 (I) and derivatives in the substrate binding pocket of human CD73 based on an X-ray structure (PDB ID: 6s7f). (A) Binding pose of the co-crystallized inhibitor PSB-12379 (orange); (B) binding pose of I (green); (C) binding pose of 31 (marine blue), and (D) binding pose of 37 (magenta). Important amino acids are colored in orange and shown in line representation. The two zinc ions in the substrate binding site are represented as blue spheres. Oxygen atoms are colored in red and nitrogen atoms in blue.

Article Snippet: The cDNAs of the human enzymes NPP1, NPP3, NPP5, CD38 and CD73 (Genbank accession no. NM_006258, NM_005021, NM_021572, NM_ 001775, and NM_002526, respectively) were obtained from Origene (Rockville, USA).

Techniques: Binding Assay

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 is CD73-specific. ( A ) 22E6 binds to HEK293 cells stably transfected with a CD73 expression plasmid (293/CD73) but not to parental HEK293 cells (293). ( B ) 22E6 specifically precipitates CD73 from lysates. ( C ) 22E6 binds to various permanent cancer cell lines and cells isolated from primary ascites. Plain histograms in ( A , C ) = isotype control antibody.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Isolation, Control

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 inhibits CD73 enzyme activity. ( A ) U138 MG glioblastoma cells were incubated with APCP, 22E6 or an isotype control antibody in the presence of AMP. The amount of inorganic phosphate was measured in a Malachite Green assay. ( B ) The IC50 of 22E6 was calculated to be 0.5 µg/mL, corresponding to approximately 3.5 nM. ( C ) The generation of inorganic phosphate from ADP is CD73-dependent and effectively blocked by 22E6 as shown on MDA-MB231 and GBM20 cells. CD73-negative T47-D do not produce detectable amounts of phosphate. Experiments were performed three times. ** p < 0.01; **** p < 0.0001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Activity Assay, Incubation, Control, Malachite Green Assay

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 blocks CD73 enzyme activity on tumor-derived extracellular vesicles. ( A ) TEVs from GBM20 cells were floated into a Optiprep gradient and separated into eight fractions, which were subsequently tested for the presence of the EV markers CD63, CD81 and TSG-101, as well as of CD73. ( B ) Particle numbers in fractions 1 to 8 were counted by NTA, and the protein content was measured in a Bradford assay. ( C ) Fraction 3 contains vesicles which stain positive for CD63, CD81 and CD73, while it is negative for Calnexin indicative for a cell-free preparation. ( D ) APCP and 22E6 block CD73 activity on TEVs from GBM20 cells and ( E ) isolated from primary ascites from a patient with ovarian cancer. ( F ) enzyme activity can be transferred by CD73-positive TEVs from GBM20 cells onto CD73-negative T47-D cells. Experiments were performed at least three times. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Activity Assay, Derivative Assay, Bradford Assay, Staining, Blocking Assay, Isolation

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 is a non-competitive inhibitor of CD73. ( A ) Incubation of U138 MG cells with 22E6 and increasing concentrations of AMP resulted in a decreases Vmax. ( B ) The 22E6 antibody but not the 22E6 Fab fragments inhibits CD73 activity. One representative experiment of three is shown. *** p < 0.001; **** p < 0.0001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Incubation, Activity Assay

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 does not inhibit enzyme activity of soluble CD73. ( A ) specific precipitation of recombinant CD73 by 22E6. ( B ) 22E6 does not detectably inhibit enzyme activity of soluble CD73. Error bars = SD; p < 0.001 for APCP, n.s. for Isotype and 22E6. This experiment was performed at least four times.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Activity Assay, Recombinant

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 induces downregulation of CD73 on PDX ALL cells in vivo. ( A ) PDX ALL cells express surface CD73 before transplantation. Black line = 22E6; tinted histogram = isotype control. ( B ) CD73 on PDX ALL cells is enzymatically active, and this activity can be inhibited with 22E6 and APCP. ( C ) NSG mice were transplanted with ALL-272 or ALL-1124 cells. Tumor burden was monitored by bioluminescence imaging (BLI). Mice were treated with 100 µg 22E9 twice per week ( n = 5 for ALL-272, n = 6 for ALL-1124; black triangles) or left untreated ( n = 3 for ALL-272, n = 4 for ALL-1124; grey dots). Tumor burden (BLI signal) relative to treatment start was calculated. Data of two independent experiments were integrated. Shown are individual mice (dots) as well as mean ± standard deviation (line). Tumor burdens of ALL-1124 between control and 22E6 treated mice differed significantly at days 35 ( p < 0.05) and 42 ( p < 0.01). At high tumor burden, mice were sacrificed, and cells were isolated. ( D ) Flow Cytometric analysis of isolated xenograft ALL cells from 22E6 treated (solid line) and control mice (dashed line) checking for expression of CD73. Black line = 22E6 anti-CD73; Tinted = control. ( E ) AMPase assay with ALL cell lines testing for enzymatic activity of CD73 of isolated xenograft ALL cells from 22E6 treated and control mice. *** p < 0.001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: In Vivo, Transplantation Assay, Control, Activity Assay, Imaging, Standard Deviation, Isolation, Expressing