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: Cancers

Article Title: A Novel Bispecific Antibody for EpCAM-Directed Inhibition of the CD73/Adenosine Immune Checkpoint in Ovarian Cancer

doi: 10.3390/cancers15143651

Figure Lengend Snippet: BsAb CD73xEpCAM has dual binding specificity for CD73 and EpCAM. ( A ) Competitive binding assay in which bsAb CD73xEpCAM (1 μg/mL) was pretreated with excess amounts of soluble CD73 (sCD73), soluble EpCAM (sEpCAM), or a combination thereof (10 μg) and then evaluated for binding to OvCAR3 cancer cells. ( B ) Dose-dependent binding of bsAb CD73xEpCAM to L37 and L37.EpCAM. ( C ) Binding of bsAb CD73xEpCAM (1 μg/mL) (or controls) to parental OvCAR3and corresponding CD73-KO and EpCAM-KO variants thereof. ( D ) Residual binding of bsAb CD73xEpCAM (1μg/mL) (or controls) to OvCAR3 cancer cells at indicated time points after 30 s of incubation and subsequent washing with PBS. ( E ) Residual CD73 membrane presence on OvCAR3 cells after treatment with bsAb CD73xEpCAM (0.01–10 µg/mL) (or controls) for 5 h. All experiments were analyzed by flow cytometry. All graphs represent mean ± SD.

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega, Madison, WI, USA) using a eukaryotic plasmid containing cDNA encoding human CD73 (Hygromycin B selection, Origene, Rockville, MD, USA).

Techniques: Binding Assay, Competitive Binding Assay, Incubation, Membrane, Flow Cytometry

Journal: Cancers

Article Title: A Novel Bispecific Antibody for EpCAM-Directed Inhibition of the CD73/Adenosine Immune Checkpoint in Ovarian Cancer

doi: 10.3390/cancers15143651

Figure Lengend Snippet: BsAb CD73xEpCAM potently inhibits the enzyme activity of CD73 in an EpCAM-directed manner. ( A ) Kinetics of inhibition of OvCAR3-exposed CD73 enzyme activity by bsAb CD73xEpCAM (1 μg/mL) (or controls). Inhibition of CD73 enzyme activity by treatment (15 min) with bsAb CD73xEpCAM (1 μg/mL) (or controls) of ( B ) CD73 pos /EpCAM pos cancer cell lines, ( C ) CD73 pos /EpCAM neg cancer cell lines, and ( D ) primary-OC-patient-derived carcinoma cells. ( E ) Competitive CD73 enzyme inhibition assay on OvCAR3 cells after treatment (15 min) with bsAb CD73xEpCAM in the presence of excess amounts of soluble EpCAM (sEpCAM). ( F ) Dose-dependent inhibition of CD73 enzyme activity by bsAb CD73xEpCAM (0.01–10 µg/mL) exposed on parental OvCAR3 cells versus OvCAR3 EpCAM-KO cells. CD73-mediated hydrolysis of AMP to ADO was evaluated using a colorimetric malachite green-based Pi assay. All graphs represent mean ± SD. Statistical analysis in ( B , D ) (group-mean) was performed using unpaired t -test (* p < 0.05, ** p < 0.01).

Article Snippet: CHO.CD73 cells stably expressing human CD73 were generated by lipofection (Fugene-HD, Promega, Madison, WI, USA) using a eukaryotic plasmid containing cDNA encoding human CD73 (Hygromycin B selection, Origene, Rockville, MD, USA).

Techniques: Activity Assay, Inhibition, Derivative Assay, Enzyme Inhibition Assay