Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1.

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Figure 8. Negative corre- lation between ecto- enzymes and CCND1 expression in CRC. (A) IHC staining of human normal colon (n ¼ 40) and colon adenocarcinoma (n ¼ 64) specimens in a tissue microarray for CCND1 and CD163 (a macrophage marker). (B) Relative intensities of CCND1 staining in the lamina propria of a normal and cancerous colon. **P < .01 (unpaired t test). (C) Correlation analysis of NT5E and CCND1 expres- sion in the TCGA colorectal adenocarcinoma dataset. ***P < .001 (Student t test).

Article Snippet: For the fluorescence-activated cell sorting analysis, cells were stained with APC,-anti human CD163 (clone: GHI/61), PD-anti human CTLA4 (clone: L3D10), Alexa 647-anti human IDO1 (clone: 2E2/IDO1), PE-anti human DR4 (clone: DJR1), FITC-anti human CD47 (clone: REA220, FITC-anti human MICA&B (clone: 6D4), PE-anti human PD-L1 (clone: MIH2), PD-anti human CD69 (clone: FNM50), FITCanti human CD2 (clone: RPA-2.10), FITC-anti human CD20 (clone: 2H7), APC-anti mouse F4/80 (clone: BM8), Brilliant Violet 421TM-anti mouse CD11b (clone: M1/70), Brilliant Violet 570TM-anti mouse CD45 (clone: 104), PE-anti-human CD45 (clone: HI30), monoclonal antibodies (Biolegend; San Diego, CA, USA), FITC-anti-human CD40 (clone: REA733), PE-anti human CD80 (clone: 2D10), PE/vio770-anti human CD206 (clone: DCN228), PE-anti human CD62E (clone: REA280), PE-anti human CD192 (clone: REA264), APC-anti human I-CAM (clone: REA266), APC-anti human HLA-DR, DP, DQ (clone: REA332), APC-anti human CCL2 (clone: REA485), APC-anti human CD14 (clone: HI30), Vioblue-anti human CD31 (clone: TUK4), FTIC-anti-human CD86 (clone: FM95), PE-anti human CD73 (clone: AD2), PE-anti mouse PD-L1 (clone: 60533), PE-anti mouse CD39, and PE-anti human CD39 (clone: REA739) monoclonal antibodies (Miltenyi Biotec; Bergisch Gladbach, Germany), and PE/CF594anti human CD3 (clone: UCHT1), PE-anti human CD44 (clone: G44-26), PE-anti human CD183 (clone: 150503), and PD-1 (clone: REA739) monoclonal antibodies (BD Biosciences; San Jose, CA, USA) following the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Marker, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Human placenta-derived mesenchymal stem cells stimulate neuronal regeneration by promoting axon growth and restoring neuronal activity

doi: 10.3389/fcell.2023.1328261

Figure Lengend Snippet: Characterization of hPMSCs. (A) Morphology at passage 3. Both normoxic and hypoxic cultures show highly homogeneous spindle-like shape. Scale bar: 500 µm (B) Proliferation capacity. Cumulative population doublings of hPMSCs grown under hypoxia (square) are higher than cultures grown in normoxia (dot). (C) Flow cytometry shows specific hMSCs marker expression pattern: positive for CD105, CD44, CD90, CD73 and negative for CD14, CD19, CD34, CD45 and HLA-DR. (D) Osteogenic differentiation was confirmed by Alizarin red staining of calcium deposits. (E) Adipogenesis differentiation was followed by Oil Red O staining of lipids vacuoles (black arrows indicate lipid droplets). (F) Alcian Blue staining of proteoglycans demonstrated chondrogenesis differentiation. Scale bar: 100 μm. Two-way ANOVA and post hoc Sidak test for multiple comparisons between means (** p ≤ 0.005; * p ≤ 0.05).

Article Snippet: Antibodies against the following human antigens were used: CD105-FITC (Miltenyi Biotect, Bergisch Gladbach, Germany, cat# 130-112-327, 1:50), CD90-FITC (Miltenyi Biotec, cat# 130-114-901, 1:50), CD44-VioBlue (Miltenyi Biotec, cat# 130-113-906, 1:50), CD73-APC (Miltenyi Biotec, cat# 130-111-909, 1:50), MSC Phenotyping Cocktail-PE (CD34, CD14, CD19, CD45, Miltenyi Biotec cat# 130-125-285, dilution according to the manufacturer’s instructions).

Techniques: Flow Cytometry, Marker, Expressing, Staining

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: Glycobiology

Article Title: Universal phosphatase-coupled glycosyltransferase assay.

doi: 10.1093/glycob/cwq187

Figure Lengend Snippet: Fig. 1. Phosphatase-coupled glycosyltransferase assay. This strategy can be applied to any glycosyltransferase reaction where the leaving group contains a removable phosphate. (A) Glycosyltransferase reaction with a diphosphonucleotide leaving group can be coupled to an ENTPD, such as CD39L3. (B) Glycosyltransferase reaction with a monophosphonucleotide leaving group can be coupled to a 5′-nucleotidase, such as CD73. The inorganic phosphate released by the coupling phosphatase may be detected using various phosphate detection reagents.

Article Snippet: Biantennary N-linked core pentasaccharide was from V-LABS, Inc. Recombinant human KTELC1, ST6GAL1, CD73, CD39L3, TNAP, MUC-1, TcdB containing the glucosyltransferase domain, other recombinant glycosyltransferases from Table II and Malachite Green Phosphate Detection Kit were from R&D Systems.

Techniques:

Journal: Glycobiology

Article Title: Universal phosphatase-coupled glycosyltransferase assay.

doi: 10.1093/glycob/cwq187

Figure Lengend Snippet: Fig. 2. Common leaving nucleotides treated with different phosphatases/nucleotidases. Common leaving nucleotides of glycosyltransferase reactions, CMP (green), GDP (purple) and UDP (blue), were treated with CD73, CD39L3 and TNAP. All reactions were done in 50 µL of 25 mM Tris, 150 mM NaCl, 5 mM MgCl2 and 5 mM MnCl2 at pH 7.5 in a 96-well plate at room temperature for 5 min. Released phosphate was detected using the Malachite Green Phosphate Detection kit. Reactions or standards with phosphate content >4 nmol were diluted to keep the absorbance within the linear range of the detection kit. The absorbances (ODs) were obtained by multiplying the observed ODs by the dilution factors. In each case, ODs were plotted against the nucleotide inputs. Phosphate standard curves were plotted with a dashed red line for comparison. Treatment with 0.1 µg of CD73 (A), 0.1 µg of CD39L3 (B), 0.1 µg of TNAP (C) or 1 µg of TNAP (D).

Article Snippet: Biantennary N-linked core pentasaccharide was from V-LABS, Inc. Recombinant human KTELC1, ST6GAL1, CD73, CD39L3, TNAP, MUC-1, TcdB containing the glucosyltransferase domain, other recombinant glycosyltransferases from Table II and Malachite Green Phosphate Detection Kit were from R&D Systems.

Techniques: Comparison

Journal: Glycobiology

Article Title: Universal phosphatase-coupled glycosyltransferase assay.

doi: 10.1093/glycob/cwq187

Figure Lengend Snippet: Fig. 5. ST6GAL1 assayed using CD73. Each reaction was coupled to 0.1 µg of CD73. The phosphate content of each well was calculated based on a phosphate standard curve determined side by side. Km and apparent Vmax (V 0 max) were obtained by fitting the data to the Michaelis–Menten equation. (A) Specific activity (SA) against donor substrate CMP-NeuAc in the presence of 1 mM acceptor LN. (B) Specific activity vs. acceptor substrate LN in the presence of 0.2 mM CMP-NeuAc. (C) Activity vs. enzyme dose in the presence of 2 mM CMP-NeuAc and 8 mM LN. The dashed line represents the linear regression line of the data points. The slope of the line represents the specific activity and was taken as the measured Vmax. R, correlation coefficient.

Article Snippet: Biantennary N-linked core pentasaccharide was from V-LABS, Inc. Recombinant human KTELC1, ST6GAL1, CD73, CD39L3, TNAP, MUC-1, TcdB containing the glucosyltransferase domain, other recombinant glycosyltransferases from Table II and Malachite Green Phosphate Detection Kit were from R&D Systems.

Techniques: Activity Assay

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet:

Article Snippet: Anti-5′-Nucleotidase Clone 4G6E3 , Novus Bio , Cat# NBP2-37271.

Techniques: Recombinant, Blocking Assay, Western Blot, cDNA Synthesis, Cell Isolation, RNA Sequencing, CRISPR, Plasmid Preparation, Software