liver specific nt5e cd73  (Anhui Medical University)

Journal: MedComm

Article Title: Both Genetic Manipulation and Acupuncture Alleviate Depressive‐Like Behavior Mediated by Astrocytic P2X7 Receptors

doi: 10.1002/mco2.71042

Figure Lengend Snippet: Treatment with EA prevents the CUMS‐induced increase in expression of P2X7Rs. Determination by quantitative (q)PCR or Western blot of mRNA and protein for P2X7Rs, the purine‐metabolizing enzymes CD39 and CD73, the exocytotic vesicular proteins SNAP23 and SNAP25, and the ATP cellular release protein CALHM2 in mPFC brain slices of mice. p2x7, cd39, cd73, snap23, snap25, calhm2, mRNAs for the respective proteins. Determinations were under control conditions in the medial prefrontal cortex (mPFC) prepared from control mice, as well as in the mPFC of mice treated with CUMS either alone or in combination with EA or sham EA. (A–F) Expression of mRNAs as measured by the ΔΔCt method with respect to β‐actin‐RNA. Relative amounts of p2x7r (A), cd39 (B), cd73 (C), snap23 (D), snap25 (E), and calhm2 (F) mRNAs. (G–L) Representative immunoblots and quantification of the proteins for P2X7R (G), CD39 (H), CD73 (I), SNAP23 (J), SNAP25 (K), and CALHM2 (L) as a ratio of the spot of glycerinaldehyd‐3‐phosphat‐dehydrogenase (GAPDH). (M) Expression of p2x7R mRNA in control mice after EA or sham EA1 (see Section ) treatment. (N) Expression of P2X7 protein in control mice after EA or sham EA1 treatment. All data are shown as mean ± SEM. One‐way ANOVA was followed by the Holm–Sidak test for statistical analysis in case of normal distribution (A–L); * p < 0.05, ** p < 0.01 compared to the control group. The number of individual animals is indicated in each column.

Article Snippet: Membranes were blocked with 5% skimmed milk in Tris‐buffered saline with Tween 20 for 1 h at room temperature and subsequently incubated overnight at 4°C with the following primary antibodies: P2X7R (1:1000; Abcam, Cambridge, UK), CD39 (1:1000; Abcam), CD73 (1:1000; Proteintech, Rosemont, USA), SNAP23 (1:1000; Proteintech), SNAP25 (1:1000; Abcam), CALHM2 (1:400; Proteintech), and GAPDH (1:1000; Proteintech).

Techniques: Expressing, Western Blot, Control, Analysis

Journal: Nature Communications

Article Title: CD73 disruption with tumor-specific CRISPR/Cas9 nanoparticles reduces adenosine-mediated immunosuppression and amplifies response to chemotherapy

doi: 10.1038/s41467-026-77547-4

Figure Lengend Snippet: a Size distribution of iRGD-DC@CaP as determined by dynamic light scattering, and morphology of iRGD-DC@CaP as revealed by TEM images. Scale bar: 200 nm. b , c DOX and Cas9 release from iRGD-DC@CaP under different situations ( n = 3 biologically independent samples). Representative flow cytometry analysis images ( d ) and quantitative analysis ( e ) of cellular uptake in B16F10 cells following a 4 h incubation with iRGD-DC@CaP or controls ( n = 3 biologically independent samples). f Confocal laser scanning microscopy images of B16F10 cells following a 4 h incubation with iRGD-DC@CaP or controls. Cas9 was labeled with Alexa Fluor 647 (AF647; red), and the nuclei were stained with DAPI (blue). Scale bars: 50 µm. T7EI assay ( g ) and quantitative analysis ( h ) of indel frequencies of the Nt5e gene in B16F10 cells transfected with iRGD-DC CD73 @CaP or controls for 48 h ( n = 3 biologically independent samples). i Mutation rates of Nt5e examined by NGS analysis in B16F10 cells treated with iRGD-DC CD73 @CaP. j , k Western blot ( j ) and flow cytometry quantitative analysis ( k ) of CD73 protein expression in B16F10 cells after 72 h incubation with iRGD-DC CD73 @CaP or controls ( n = 3 biologically independent samples). l The ADO contents in the cell medium of B16F10 cells after 72 h incubation with iRGD-DC CD73 @CaP or controls detected by ELISA kits ( n = 3 biologically independent samples). The experiments for f and i were independently repeated three times with similar results. Data is presented as the mean ± SD. Statistical difference was assessed in ( e , h , j , k and l ) using one-way ANOVA with Dunnett’s multiple comparison test by comparing the iRGD-DC CD73 @CaP group with each of the other treatment groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. G1: saline (negative control); G2: Free Cas9 RNP; G3: iRGD-modified nanoparticles loaded with scrambled sgRNA (iRGD-DC Scr @CaP, negative control); G4: Lipofectamine™ CRISPRMAX™-mediated delivery of Cas9/sgCD73 (MAX Cas9/sgCD73, positive control); G5: nanoparticle without iRGD loaded with Cas9/sgCD73 (DC CD73 @CaP); G6: iRGD-modified nanoparticle loaded with Cas9/sgCD73 (iRGD-DC CD73 @CaP). The exact P value and Source data are provided as a Source Data file.

Article Snippet: The blocked PVDF membrane using skim milk was incubated with primary antibody against CD73 (12231-1-AP, Proteintech) at a dilution of 1:2000 overnight at 4 °C.

Techniques: Dynamic Light Scattering Assay, Morphology, Flow Cytometry, Analysis, Incubation, Confocal, Laser-Scanning Microscopy, Labeling, Staining, T7EI Assay, Transfection, Mutagenesis, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Comparison, Saline, Negative Control, Modification, Positive Control

Journal: Nature Communications

Article Title: CD73 disruption with tumor-specific CRISPR/Cas9 nanoparticles reduces adenosine-mediated immunosuppression and amplifies response to chemotherapy

doi: 10.1038/s41467-026-77547-4

Figure Lengend Snippet: a Cytotoxicity effects of B16F10 cells after receiving different treatments for 72 h ( n = 5 biologically independent samples). b The apoptosis rates of B16F10 cells detected by flow cytometry after different treatments for 72 h ( n = 3 biologically independent samples). c Western blotting analysis of HMGB1 and CRT protein expression in B16F10 cells after receiving different treatments for 72 h ( n = 3 biologically independent samples). d Immunofluorescence analysis of CRT (green) and HMGB1 (green) after receiving different treatments in B16F10 cells for 72 h. The nucleus was stained using DAPI (blue). Scale bar: 20 μm. Quantitative analysis of e CRT and f HMGB1 expression detected by CLSM in B16F10 cells receiving different treatments using ImageJ ( n = 5 randomly selected fields of view). The experiments in ( e , f ) were repeated independently three times with similar results. g HMGB1 release measured by ELISA kits after receiving different treatments for 72 h ( n = 5 biologically independent samples). h The ATP release levels after receiving different treatments for 72 h ( n = 3 biologically independent samples). Data is presented as the mean ± SD. Statistical differences were assessed in ( a , b , e , f , g and h ) using one-way ANOVA with Dunnett’s multiple comparison test by comparing the iRGD-DC CD73 @CaP group with each of the other treatment groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. G1: saline (negative control); G2: Free Cas9 RNP; G3: iRGD-modified nanoparticles loaded with scrambled sgRNA (iRGD-DC Scr @CaP, negative control); G4: nanoparticle without iRGD loaded with Cas9/sgCD73 (DC CD73 @CaP); G5: iRGD-modified nanoparticle loaded with Cas9/ sgCD73 (iRGD-DC CD73 @CaP). The exact P value and Source data are provided as a Source Data file.

Article Snippet: The blocked PVDF membrane using skim milk was incubated with primary antibody against CD73 (12231-1-AP, Proteintech) at a dilution of 1:2000 overnight at 4 °C.

Techniques: Flow Cytometry, Western Blot, Analysis, Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Comparison, Saline, Negative Control, Modification

Journal: Nature Communications

Article Title: CD73 disruption with tumor-specific CRISPR/Cas9 nanoparticles reduces adenosine-mediated immunosuppression and amplifies response to chemotherapy

doi: 10.1038/s41467-026-77547-4

Figure Lengend Snippet: a Fluorescence imaging of B16F10 tumor-bearing C57BL/6 mice following injection of iRGD-DC@CaP and controls ( n = 3 mice). b Quantitative analysis of AF647-Cas9 fluorescence from major organs in mice bearing B16F10 at 1 h after intravenous injection of iRGD-DC@CaP or controls ( n = 3 mice). “% ID/g” represents the percentage of the injected dose per gram of tissue. The exact P value: DC@CaP vs. iRGD-DC@CaP ** P = 0.0087. c Schematic illustration of B16F10 tumor therapy in vivo. Created in BioRender. Sun, X. (2026) https://BioRender.com/f4h4ymp . d Individual and average tumor growth curves of B16F10 following different treatments ( n = 5 mice). e Tumor weight and the tumor inhibition ratio following different treatments ( n = 5 mice). f Survival analysis of the mice following different treatments ( n = 6 mice). P values were determined by the log-rank test. g Indel frequency of Nt5e loci measured by T7EI assay in B16F10 tumor tissues with the indicated treatments. The experiment was independently repeated three times with similar results. h Western blotting and relative quantitative analysis of CD73 proteins in B16F10 tumor tissues with the indicated treatments ( n = 3 biologically independent samples). i The flow cytometry quantitative analysis of the CD73 expression on tumor cells (CD45 − ), endothelial cells (CD45 − CD31 + ), cancer-associated fibroblasts (CAFs) (CD45 − CD31 − GP38 + ), macrophages (CD45 + CD11b + F4/80 + ), DCs (CD45 + CD11c + MHC-II + ), Tregs (CD45 + CD4 + CD25 + Foxp3 + ) and MDSCs (CD45 + CD11b + Gr-1 + ) in B16F10 tumor tissues after indicated treatments ( n = 5 mice). j The ADO contents in the tumor after different treatments ( n = 5 mice). Data is presented as the mean ± SD. Statistical differences were assessed using one-way ANOVA in ( b , e , h , i and j ) or two-way ANOVA in ( d ) with Dunnett’s multiple comparison test by comparing the iRGD-DC CD73 @CaP group with each of the other treatment groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. G1: saline (negative control); G2: iRGD-modified nanoparticles loaded with scrambled sgRNA (iRGD-DC Scr @CaP, negative control); G3: iRGD-modified nanoparticle loaded with Cas9/sgCD73 (iRGD-DC CD73 @CaP). The exact P value and Source data are provided as a Source Data file.

Article Snippet: The blocked PVDF membrane using skim milk was incubated with primary antibody against CD73 (12231-1-AP, Proteintech) at a dilution of 1:2000 overnight at 4 °C.

Techniques: Fluorescence, Imaging, Injection, Analysis, Mouse Assay, Tissue, In Vivo, Inhibition, T7EI Assay, Western Blot, Flow Cytometry, Expressing, Comparison, Saline, Negative Control, Modification

Journal: Nature Communications

Article Title: CD73 disruption with tumor-specific CRISPR/Cas9 nanoparticles reduces adenosine-mediated immunosuppression and amplifies response to chemotherapy

doi: 10.1038/s41467-026-77547-4

Figure Lengend Snippet: a Flow cytometry quantitative analysis of CD80 + CD86 + DCs in the tumor after different treatments ( n = 5 mice). b Flow cytometry quantitative analysis of the absolute number of CD8 + T cells in tumors ( n = 5 mice). c Flow cytometry quantitative analysis of CD3 + CD8 + T cells in the tumor after different treatments ( n = 5 mice). d Flow cytometry quantitative analysis of CD4 + CD25 + Foxp3 + Tregs cells in the tumor after different treatments ( n = 5 mice). e The ratio of CD8 + T cells/Tregs in the tumor after different treatments ( n = 5 mice). Flow cytometry quantitative analysis of CD8 + GranzymeB + ( f ), CD8 + IFNγ + ( g ), CD8 + TNFα + ( h ), CD8 + IL2 + ( i ), CD8 + CD69 + ( j ), CD8 + PD1 + ( k ), and CD8 + TIM-3 + ( l ) T cells in the tumor after different treatments ( n = 5 mice). m Flow cytometry quantitative analysis of CD8 + CD44 + T cells in spleen after different treatments ( n = 5 mice). n Flow cytometry quantitative analysis of CD3 − CD49b + NK cells in the tumor after different treatments ( n = 5 mice). o Flow cytometry quantitative analysis of F4/80 + CD86 + cells in tumor after different treatments ( n = 5 mice). p Flow cytometry quantitative analysis of F4/80 + iNOS + cells in tumor after different treatments ( n = 5 mice). q Flow cytometry quantitative analysis of F4/80 + CD206 + cells in tumor after different treatments ( n = 5 mice). r Flow cytometry quantitative analysis of F4/80 + Arg1 + cells in tumor after different treatments ( n = 5 mice). s The ratio of F4/80 + CD86 + M1-like cells versus F4/80 + CD206 + M2-like cells in tumor after different treatments ( n = 5 mice). t Flow cytometry quantitative analysis of CD11b + Gr-1 + MDSCs in the tumor after different treatments ( n = 5 mice). Data is presented as the mean ± SD. Statistical differences were assessed in a–t using one-way ANOVA with Dunnett’s multiple comparison test by comparing the iRGD-DC CD73 @CaP group with each of the other treatment groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. G1: saline (negative control); G2: iRGD-modified nanoparticles loaded with scrambled sgRNA (iRGD-DC Scr @CaP, negative control); G3: iRGD-modified nanoparticle loaded with Cas9/sgCD73 (iRGD-DC CD73 @CaP). The exact P value and Source data are provided as a Source Data file.

Article Snippet: The blocked PVDF membrane using skim milk was incubated with primary antibody against CD73 (12231-1-AP, Proteintech) at a dilution of 1:2000 overnight at 4 °C.

Techniques: Flow Cytometry, Analysis, Comparison, Saline, Negative Control, Modification

Journal: Nature Communications

Article Title: CD73 disruption with tumor-specific CRISPR/Cas9 nanoparticles reduces adenosine-mediated immunosuppression and amplifies response to chemotherapy

doi: 10.1038/s41467-026-77547-4

Figure Lengend Snippet: a Volcano plot of differentially expressed genes (DEGs) in B16F10 tumor tissue treated with iRGD-DC CD73 @CaP compared to the saline group ( n = 3 mice). Statistical significance was assessed using two-sided tests, and P values were adjusted for multiple testing using the Benjamini–Hochberg method. Genes with |log2FoldChange| ≥ 1 and adjusted P < 0.05 were considered differentially expressed. b Heatmap of selected differentially expressed genes (DEGs) in B16F10 tumor tissue treated with iRGD-DC CD73 @CaP compared to saline group ( n = 3 mice). Red: upregulation; blue: downregulation. c Enriched gene ontology (GO) terms related to DEGs associated with immune activation ( n = 3 mice). d KEGG enrichment analysis of DEGs after iRGD-DC CD73 @CaP treatment ( n = 3 mice). GO and KEGG enrichment analyses were performed using a hypergeometric test. P values were adjusted for multiple comparisons using the Benjamini–Hochberg method. e GSEA enrichment analysis indicating upregulated pathways associated with antitumor immune response after iRGD-DC CD73 @CaP treatment ( n = 3 mice). Source data are provided as a Source Data file.

Article Snippet: The blocked PVDF membrane using skim milk was incubated with primary antibody against CD73 (12231-1-AP, Proteintech) at a dilution of 1:2000 overnight at 4 °C.

Techniques: Tissue, Saline, Activation Assay, Analysis

Journal: Nature Communications

Article Title: CD73 disruption with tumor-specific CRISPR/Cas9 nanoparticles reduces adenosine-mediated immunosuppression and amplifies response to chemotherapy

doi: 10.1038/s41467-026-77547-4

Figure Lengend Snippet: a Schematic illustration of experimental design for 4T1 tumor therapy in vivo. b , c Average ( b ) and individual ( c ) tumor growth curves of 4T1 following different treatments ( n = 5 mice). d Tumor weight and tumor inhibition ratio following different treatments ( n = 5 mice). e Survival analysis of the mice following different treatments ( n = 6 mice). f Indel frequency of Nt5e loci measured by T7EI assay in 4T1 tumor tissues with the indicated treatments. The experiment was independently repeated three times with similar results. g Western blot and relative quantitative analysis of CD73 protein expression in 4T1 tumor tissues with the indicated treatments ( n = 3 biologically independent samples). h The ADO contents in the tumor after different treatments ( n = 5 mice). i–t Flow cytometry quantitative analysis of CD80 + CD86 + DCs ( i ), absolute number of CD8 + T cells ( j ), CD3 + CD8 + T cells ( k ), CD4 + CD25 + Foxp3 + Tregs ( l ), CD8 + T/Tregs ratio ( m ), CD3 − CD49b + NK cells ( n ), F4/80 + CD86 + cells ( o ), F4/80 + iNOS + cells ( p ), F4/80 + CD206 + cells ( q ), F4/80 + Arg1 + cells ( r ), F4/80 + CD86 + M1/F4/80 + CD206 + M2 ratio ( s ), and CD11b + Gr-1 + MDSCs ( t ) in tumor after different treatments ( n = 5 mice). u–w Contents of the IL-2 ( u ), TNF-α ( v ), and IFN-γ ( w ) in tumor tissues after different treatments ( n = 5 mice). Data is presented as the mean ± SD. Statistical differences were analyzed by two-way ANOVA with Dunnett’s multiple comparison test in ( b ), one-way ANOVA with Dunnett’s multiple comparison test in ( d , g , and h–w ) by comparing the iRGD-DC CD73 @CaP group with each of the other treatment groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. G1: saline (negative control); G2: iRGD-modified nanoparticles loaded with scrambled sgRNA (iRGD-DC Scr @CaP, negative control); G3: iRGD-modified nanoparticle loaded with Cas9/sgCD73 (iRGD-DC CD73 @CaP). The exact P value and Source data are provided as a Source Data file.

Article Snippet: The blocked PVDF membrane using skim milk was incubated with primary antibody against CD73 (12231-1-AP, Proteintech) at a dilution of 1:2000 overnight at 4 °C.

Techniques: In Vivo, Inhibition, Analysis, T7EI Assay, Western Blot, Expressing, Flow Cytometry, Comparison, Saline, Negative Control, Modification