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: For immunocytochemical costaining, cells were incubated with primary antibody against CD39 (1:100; ab227840, Abcam, Cambridge, UK) and CD73 (5 μg/ml; AF4488, R&D Systems) and stained with secondary antibody using anti-donkey Alexa Fluor 488 and anti-goat Alexa Fluor 647 (1:250; Jackson ImmunoResearch).

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: For immunocytochemical costaining, cells were incubated with primary antibody against CD39 (1:100; ab227840, Abcam, Cambridge, UK) and CD73 (5 μg/ml; AF4488, R&D Systems) and stained with secondary antibody using anti-donkey Alexa Fluor 488 and anti-goat Alexa Fluor 647 (1:250; Jackson ImmunoResearch).

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: For immunocytochemical costaining, cells were incubated with primary antibody against CD39 (1:100; ab227840, Abcam, Cambridge, UK) and CD73 (5 μg/ml; AF4488, R&D Systems) and stained with secondary antibody using anti-donkey Alexa Fluor 488 and anti-goat Alexa Fluor 647 (1:250; Jackson ImmunoResearch).

Techniques: Knockdown, In Vitro, Control, Concentration 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 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