Journal: Frontiers in immunology

Article Title: Immunometabolic interference between cancer and COVID-19.

doi: 10.3389/fimmu.2023.1168455

Figure Lengend Snippet: FIGURE 9 Flow cytometry analysis of NAMPT and NAD-dependent enzymes in the different patient cohorts. Box-and-whisker plots representing FACS quantification of (A) NAMPT, (B) pSIRT1, (C) CD38, (D) CD39 and (E) PARP expression in the indicated immune cell populations (CD3+ T cells, CD19+

Article Snippet: Cells were incubated with the following antihuman antibodies for 20 min at 4°C: CD33 PerCP Cy5.5 (WM53) (0.6:100, Biolegend cat No. 303402), CD14 BV650 (M5E2) (1.25:100, Biolegend cat. No. 301835), CD16 BV711 (3G8) (0.3:100, Biolegend cat. No. 302044), HLA-DR BV421 (L243) (1.25:100, Biolegend Cat No. 307636), CD15 BV786 (HI98) (1.25:100, BD Biosciences cat. No. 563838), CD11c BUV661 (B-ly6) (0.6:100, BD Biosciences cat. No. 612968), CD123 BUV395 (7G3) (0.6:100, BD Biosciences cat. No. 564195), CD38 PE-Cy7 (HIT2) (1.25:100, Invitrogen cat. No. 25- 0389-42), CD39 PE (MZ18-23C8) (0.6:100, MACSMiltenyi Biotec Inc. cat. No.130-118-668), CD39 FITC (MZ18-23C8) (1.25:100, MACS Miltenyi Biotec Inc. cat. No. 130-125-113), CD3 BV650 (OKT3) (1.25:100, Biolegend cat. No.317324), CD4 BV570 (OKT4) (2.5:100, Frontiers in Immunology 03 Biolegend cat. No. 317445), CD8 BV786 (RPA-T8) (0.6:100, BD Biosciences cat. No. 557085), CD19 AF-700 (HIB19) (2.5:100, BD Biosciences cat. No. 561031), CD45RA APC-Cy7 (HI100) (1.25:100, BD Biosciences cat. No. 560674), CD45RO BUV395 (UCHL1) (1.25:100, BD Biosciences 564292), CD27 PE-Cy5 (O323) (1.25:100, Invitrogen cat. No. 15295964), CD16 BV605 (3G8) (0.6:100, Biolegend 302040), CD3 BUV496 (UCHT1) (1.25:100, BD Biosciences cat. No. 612940), CD66b PE-Cy7(G10F5) (0.6:100 Biolegend cat. No. 305116) and IgD FITC (IA6-2) (1.25:100, Invitrogen cat. No. 11-9868-42).

Techniques: Flow Cytometry, Whisker Assay, Expressing

Journal: Technology in Cancer Research & Treatment

Article Title: B7-H3 and CD39 Co-Localization in Gastric Cancer: A Potential Prognostic Biomarker and Potential Dual-Target for Immunotherapy

doi: 10.1177/15330338251380957

Figure Lengend Snippet: Expression Patterns of B7-H3 and CD39 in Gastric Precancerous Lesions and Gastric Cancer Tissues. (A) Representative IHC Expression Patterns of B7-H3 and CD39 Across Different Pathological Stages (CSG, CAG, LGIN, HGIN, and GC). (B) Quantitative Analysis of Positive Expression Areas of CD39. (C) Quantitative Analysis of Positive Expression Areas of B7-H3. (D) Representative Multiplex IHC Expression Patterns of B7-H3 and CD39 Across Different Pathological Stages (CSG, CAG, LGIN, HGIN, and GC). (E) Quantitative Analysis of Multiplex IHC Expression of B7-H3 and CD39. (F) Representative Co-Localization Expression of B7-H3 and CD39 in GC. IHC Scale Bars: 50 µm; mIHC Scale Bars: 100 µm.

Article Snippet: The following primary antibodies were used according to the manufacturer's instructions: mouse anti-human B7-H3 monoclonal antibody, 1/200 dilution (Cat No: 66 481-1-Ig, Proteintech, China), rabbit anti-human CD39 polyclonal antibody, 1/1000 dilution (Cat No: 14211-1-AP, Proteintech, China) and mouse anti-human CD8 monoclonal antibody, 1/10 000 dilution (Cat No: 66868-1-Ig, Proteintech, China).

Techniques: Expressing, Multiplex Assay

Journal: Technology in Cancer Research & Treatment

Article Title: B7-H3 and CD39 Co-Localization in Gastric Cancer: A Potential Prognostic Biomarker and Potential Dual-Target for Immunotherapy

doi: 10.1177/15330338251380957

Figure Lengend Snippet: Co-Localization of B7-H3 and CD39 in Gastric Cancer Cells Indicates Poor Prognosis. (A, B) Representative Immunohistochemical Images Showing Low and High Expression of B7-H3 (A) and CD39 (B) in Gastric Cancer (GC) Specimens. (C) Correlation Analysis of B7-H3 and CD39 Expression. (F, G, H, I) Kaplan-Meier Survival Curves for Overall Survival of GC Patients Based on the Expression Status of B7-H3 (F), CD39 (G), Dual High Expression of B7-H3 and CD39 (H), and Co-Localized Expression Status of B7-H3-CD39 (I). Scale Bars: 100 µm.

Article Snippet: The following primary antibodies were used according to the manufacturer's instructions: mouse anti-human B7-H3 monoclonal antibody, 1/200 dilution (Cat No: 66 481-1-Ig, Proteintech, China), rabbit anti-human CD39 polyclonal antibody, 1/1000 dilution (Cat No: 14211-1-AP, Proteintech, China) and mouse anti-human CD8 monoclonal antibody, 1/10 000 dilution (Cat No: 66868-1-Ig, Proteintech, China).

Techniques: Immunohistochemical staining, Expressing

Journal: Technology in Cancer Research & Treatment

Article Title: B7-H3 and CD39 Co-Localization in Gastric Cancer: A Potential Prognostic Biomarker and Potential Dual-Target for Immunotherapy

doi: 10.1177/15330338251380957

Figure Lengend Snippet: Absence of Correlation Between Co-localization of B7-H3 and CD39 Expression and CD8 + T Cell Infiltration in Gastric Cancer. (A, B) Representative Immunohistochemical Images Showing the Expression of B7-H3 with CD8 (A) or CD39 with CD8 (B) in the Same Patient. (C) Correlation Between B7-H3 Expression and CD8 Expression, and Between CD39 Expression and CD8 Expression. (D, E) Representative Multiplex Immunohistochemistry Images of B7-H3, CD39, and CD8 (D), and Correlation Analysis Between the Co-Localization Expression Score of B7-H3 and CD39 and the Extent of CD8 Infiltration (E). (F, G) Representative Multiplex Immunohistochemistry Images of CD39 and CD8, with Arrows Indicating CD39 + CD8 + T Cells (F), and the Proportion of CD39 + CD8 + T Cells within the CD8 + T Cell Population (G). Scale Bars: 100 µm.

Article Snippet: The following primary antibodies were used according to the manufacturer's instructions: mouse anti-human B7-H3 monoclonal antibody, 1/200 dilution (Cat No: 66 481-1-Ig, Proteintech, China), rabbit anti-human CD39 polyclonal antibody, 1/1000 dilution (Cat No: 14211-1-AP, Proteintech, China) and mouse anti-human CD8 monoclonal antibody, 1/10 000 dilution (Cat No: 66868-1-Ig, Proteintech, China).

Techniques: Expressing, Immunohistochemical staining, Multiplex Assay, Immunohistochemistry

Journal: Technology in Cancer Research & Treatment

Article Title: B7-H3 and CD39 Co-Localization in Gastric Cancer: A Potential Prognostic Biomarker and Potential Dual-Target for Immunotherapy

doi: 10.1177/15330338251380957

Figure Lengend Snippet: Kaplan-Meier Survival Curves for Gastric Cancer Patients Based on Specific Expression Statuses and Immune Cell Infiltration Levels. (A, B, C, D) Kaplan-Meier Survival Curves for GC Patients Stratified by B7-H3 High CD8 Low (A) Expression Status 、 CD39 High CD8 Low (B) Expression Status 、 B7-H3-CD39 Both high CD8 Low Expression Status (C) and B7-H3-CD39 Co-localization CD8 low Expression Status (D). (E, F) Kaplan-Meier Survival Curves for GC Patients Based on CD8 + T Cell Infiltration Levels (E) and CD39 + CD8 + T Cell Infiltration Status (F).

Article Snippet: The following primary antibodies were used according to the manufacturer's instructions: mouse anti-human B7-H3 monoclonal antibody, 1/200 dilution (Cat No: 66 481-1-Ig, Proteintech, China), rabbit anti-human CD39 polyclonal antibody, 1/1000 dilution (Cat No: 14211-1-AP, Proteintech, China) and mouse anti-human CD8 monoclonal antibody, 1/10 000 dilution (Cat No: 66868-1-Ig, Proteintech, China).

Techniques: Expressing

Journal: Cancer immunology research

Article Title: CD73 Inhibits cGAS-STING and Cooperates with CD39 to Promote Pancreatic Cancer.

doi: 10.1158/2326-6066.CIR-22-0260

Figure Lengend Snippet: Figure 1. CD73 gene expression is associated with poor PDAC prognosis. (A) Meta-analysis of CD73 gene expression (median) with 5-year overall survival (5-y OS). Forest plot displays the log hazard ratios (logHR) and 95% confidence intervals (CI). Horizontal bars represent the 95% CI of effect-size. Blue diamond represents the overall effect in all PDAC patients. (B) Association between CD73 gene expression (median) and OS in CD39-High PDAC (CD73 Low: n=37; CD73 High: n=36) and CD39-Low PDAC (CD73 Low: n=37; CD73 High: n=36) from the TCGA cohort. (C) Spearman correlation heatmap between ENTPD1 (CD39), NT5E (CD73), PDCD1 (PD-1), LAG3,

Article Snippet: For CD39 staining, slides were exposed to ER1 solution (Leica cat# AR9961) for 10 minutes at 100°C, followed by a 1-hour incubation with rabbit monoclonal anti-mouse CD39 (Cell Signaling Technology, clone E2X6B, CST #14481) at room temperature at 1:100 dilution.

Techniques: Gene Expression

Journal: Cancer immunology research

Article Title: CD73 Inhibits cGAS-STING and Cooperates with CD39 to Promote Pancreatic Cancer.

doi: 10.1158/2326-6066.CIR-22-0260

Figure Lengend Snippet: Figure 2. Tumor CD73 and stromal CD39 protein expression associate with poor PDAC prognosis and suppressed immune surveillance. (A-B) Representative immunofluorescence staining of CD73 expression in epithelium (A) and CD39 expression in stroma (B) of PDAC tumors. Epithelium was determined as the compartment expressing cytokeratin (CK, green). Stroma is the CK-negative compartment. CD73, red; DAPI, blue. (C-D) Mean fluorescence intensity (MFI) of epithelial and stromal CD73 (C) and CD39 (D) expression in 104 normal adjacent pancreas (N) versus intratumoral compartment (IT) (red bars indicate mean). (E-F) Association between CD73

Article Snippet: For CD39 staining, slides were exposed to ER1 solution (Leica cat# AR9961) for 10 minutes at 100°C, followed by a 1-hour incubation with rabbit monoclonal anti-mouse CD39 (Cell Signaling Technology, clone E2X6B, CST #14481) at room temperature at 1:100 dilution.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Cancer immunology research

Article Title: CD73 Inhibits cGAS-STING and Cooperates with CD39 to Promote Pancreatic Cancer.

doi: 10.1158/2326-6066.CIR-22-0260

Figure Lengend Snippet: Figure 4. CD39 cooperates with CD73 to promote mouse PDAC. (A) KPC tumor cells were injected s.c. into CD39fl/fl LysMCre−/− mice and CD39fl/fl

Article Snippet: For CD39 staining, slides were exposed to ER1 solution (Leica cat# AR9961) for 10 minutes at 100°C, followed by a 1-hour incubation with rabbit monoclonal anti-mouse CD39 (Cell Signaling Technology, clone E2X6B, CST #14481) at room temperature at 1:100 dilution.

Techniques: Injection

Journal:

Article Title: SP1-dependent induction of CD39 facilitates hepatic ischemic preconditioning

doi: 10.4049/jimmunol.0901851

Figure Lengend Snippet: CD39 is induced by liver IP. (A) After 3 cycles of IP [5 min ischemia (white), 5 min reperfusion (black)] and 180 min reperfusion the median lobe of the murine liver was excised. (B) Total RNA was isolated from wild-type (WT) or CD39−/− mice and CD39 mRNA was determined by real-time PCR. Data were calculated relative to β-actin and expressed as fold change in transcript relative to housekeeping gene β-actin. Results are expressed as the mean ± SD of 3 mice/group. *p<0.05 significantly higher vs. WT sham. #p<0.05 significantly lower vs. WT sham. (C) The median liver lobe from wild-type (WT) or CD39−/− mice was sectioned and stained using CD39 antibody. WT samples were also stained using isotype control antibody. Note enhanced staining for CD39 in WT mice following IP treatment, particularly in the hepatocytes (inset).

Article Snippet: Immunohistochemical staining was performed with a polyclonal goat anti-mouse IgG antibody against CD39 (sc-33558 rabbit polyclonal IgG, Santa Cruz, Heidelberg, Germany) or using a negative control rabbit immunoglobulin fraction (DakoCytomation, Glostrup, Denmark).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Staining, Control

Journal:

Article Title: SP1-dependent induction of CD39 facilitates hepatic ischemic preconditioning

doi: 10.4049/jimmunol.0901851

Figure Lengend Snippet: CD39 inhibition abolishes liver protection by IP. Injury was measured after WT mice were pre-treated with the CD39 inhibitor POM-1 (3 mg/kg/h, i.a.) or saline prior to IR (IR) with or without prior IP (IP). Injury was measured for the following parameters: (A) Total RNA was isolated and CD39 mRNA was determined by real-time PCR. Data were calculated relative to β-actin and expressed as fold change in transcript relative to housekeeping gene β-actin. Results are expressed as the mean ± SD of 3 mice/group. *p<0.05 vs. WT sham. (B) LDH, (C) AST, and (D) ALT. Results are expressed as the mean ± SD of 4–8 mice/group. *p<0.05 vs. sham. #p<0.05 vs. IR+vehicle.

Article Snippet: Immunohistochemical staining was performed with a polyclonal goat anti-mouse IgG antibody against CD39 (sc-33558 rabbit polyclonal IgG, Santa Cruz, Heidelberg, Germany) or using a negative control rabbit immunoglobulin fraction (DakoCytomation, Glostrup, Denmark).

Techniques: Inhibition, Saline, Isolation, Real-time Polymerase Chain Reaction

Journal:

Article Title: SP1-dependent induction of CD39 facilitates hepatic ischemic preconditioning

doi: 10.4049/jimmunol.0901851

Figure Lengend Snippet: POM-1 abolishes liver protection by IP. (A) H&E staining of liver sections and (B) quantification of ischemic injury following treatment with the CD39 inhibitor POM-1 (3 mg/kg/h, i.a.) or saline prior to IR (IR) with or without prior IP (IP) (n=3–6 mice/group expressed as mean ± SEM). *p<0.05 vs. respective IR group.

Article Snippet: Immunohistochemical staining was performed with a polyclonal goat anti-mouse IgG antibody against CD39 (sc-33558 rabbit polyclonal IgG, Santa Cruz, Heidelberg, Germany) or using a negative control rabbit immunoglobulin fraction (DakoCytomation, Glostrup, Denmark).

Techniques: Staining, Saline

Journal:

Article Title: SP1-dependent induction of CD39 facilitates hepatic ischemic preconditioning

doi: 10.4049/jimmunol.0901851

Figure Lengend Snippet: Liver protection by IP is abolished in CD39−/− mice. CD39 deficient (CD39−/−) or WT mice were subjected to IR or IP preceding IR (IP). Sham mice underwent the same surgical procedure but without IP or IR. (A) LDH, (B) AST, and (C) ALT. Results are expressed as the mean ± SD of 4–8 mice/group. *p<0.05 vs. respective sham. #p<0.05 vs. respective IR group. (D) Hepatic adenosine concentrations with IP are attenuated in CD39−/− mice. CD39−/− or WT mice were subjected to IP. The liver was snap frozen after the last cycle of IP. Results are expressed as the mean ± SEM of 5 mice/group. *p<0.05 vs. WT+IP.

Article Snippet: Immunohistochemical staining was performed with a polyclonal goat anti-mouse IgG antibody against CD39 (sc-33558 rabbit polyclonal IgG, Santa Cruz, Heidelberg, Germany) or using a negative control rabbit immunoglobulin fraction (DakoCytomation, Glostrup, Denmark).

Techniques:

Journal:

Article Title: SP1-dependent induction of CD39 facilitates hepatic ischemic preconditioning

doi: 10.4049/jimmunol.0901851

Figure Lengend Snippet: Targeted gene-deletion of CD39 abolished hepatic protection by IP. (A) H&E staining of liver sections and (B) quantification of ischemic injury (n=3–7 mice/group expressed as mean ± SEM). *p<0.05 vs. respective IR group.

Article Snippet: Immunohistochemical staining was performed with a polyclonal goat anti-mouse IgG antibody against CD39 (sc-33558 rabbit polyclonal IgG, Santa Cruz, Heidelberg, Germany) or using a negative control rabbit immunoglobulin fraction (DakoCytomation, Glostrup, Denmark).

Techniques: Staining

Journal:

Article Title: SP1-dependent induction of CD39 facilitates hepatic ischemic preconditioning

doi: 10.4049/jimmunol.0901851

Figure Lengend Snippet: Reconstitution of CD39−/− mice with AMP is protective against IR injury. CD39−/− mice were treated with saline (vehicle) or AMP (100 µl/h of 4 mg/kg, i.a.) and subjected to IR with or without prior IP treatment. (A) LDH, (B) AST, and (C) ALT. Results are expressed as the mean ± SEM of 3–8 mice/group. *p<0.05 vs. sham. #p<0.05 vs. IR+vehicle.

Article Snippet: Immunohistochemical staining was performed with a polyclonal goat anti-mouse IgG antibody against CD39 (sc-33558 rabbit polyclonal IgG, Santa Cruz, Heidelberg, Germany) or using a negative control rabbit immunoglobulin fraction (DakoCytomation, Glostrup, Denmark).

Techniques: Saline

Journal:

Article Title: SP1-dependent induction of CD39 facilitates hepatic ischemic preconditioning

doi: 10.4049/jimmunol.0901851

Figure Lengend Snippet: Sp1 siRNA decreases SP1 and CD39 in the liver. Wild-type (WT) mice were treated with nonsense (NS siRNA) or Sp1 siRNA in combination with transfection reagent (2 mg/kg). (A) Total RNA was isolated and Sp1 transcript levels were determined by real-time PCR. Data were calculated relative to β-actin. Results are expressed as the mean ± SEM of 3 mice/group. *p<0.05 vs. NS siRNA. (B) Proteins were resolved by SDS-PAGE, transferred to nitrocellulose and probed with anti-Sp1 antibody and re-probed for β-actin. (C) Sp1 protein was calculated by densitometry relative to β-actin. (D) Total RNA was isolated and CD39 transcript levels were determined by real-time PCR. Data were calculated relative to β-actin. Results are expressed as the mean ± SEM of 3 mice/group. *p<0.05 vs. NS siRNA.

Article Snippet: Immunohistochemical staining was performed with a polyclonal goat anti-mouse IgG antibody against CD39 (sc-33558 rabbit polyclonal IgG, Santa Cruz, Heidelberg, Germany) or using a negative control rabbit immunoglobulin fraction (DakoCytomation, Glostrup, Denmark).

Techniques: Transfection, Isolation, Real-time Polymerase Chain Reaction, SDS Page

Journal:

Article Title: SP1-dependent induction of CD39 facilitates hepatic ischemic preconditioning

doi: 10.4049/jimmunol.0901851

Figure Lengend Snippet: Sp1 siRNA decreases SP1 and CD39 in the liver and inhibits injury. Wild-type (WT) mice were treated with nonsense (NS siRNA) or Sp1 siRNA in combination with transfection reagent (2 mg/kg). (A) LDH and (B) AST . Results are expressed as the mean ± SEM of 4 mice/group. *p<0.05 vs. IP-Sp1.

Article Snippet: Immunohistochemical staining was performed with a polyclonal goat anti-mouse IgG antibody against CD39 (sc-33558 rabbit polyclonal IgG, Santa Cruz, Heidelberg, Germany) or using a negative control rabbit immunoglobulin fraction (DakoCytomation, Glostrup, Denmark).

Techniques: Transfection

Journal: International journal of molecular medicine

Article Title: Apyrase protects against allergic airway inflammation by decreasing the chemotactic migration of dendritic cells in mice.

doi: 10.3892/ijmm.2014.1771

Figure Lengend Snippet: Figure 1. Ovalbumin (OVA)-induced airway inflammation leads to a reduction in CD39 expression. (A) Representative micrographs of immunofluorescence of CD39 in the lungs from the control and asthmatic (asthma) mice are shown. CD39 was detected with Rhodamine-labeled secondary antibody and located at the cytomembrane and cytoplasm of bronchial epithelial cells. Control: PBS sensitization and challenge; asthma, OVA sensitization and challenge. (B) CD39 mRNA expression was significantly reduced in the lungs from asthmatic mice compared to control mice. (C) CD39 protein expression was significantly reduced in the lungs from asthma mice compared to control mice. One representative experiment of 3 is shown. The data are presented as the means ± SD, (n=4-6 mice per group). **P<0.01 and *P<0.05; original magnification, x200.

Article Snippet: Briefly, 5-μm-thick sections were incubated with a rabbit anti-mouse CD39 antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4̊C.

Techniques: Expressing, Immunofluorescence, Control, Labeling

Journal: Cell Death & Disease

Article Title: Cancer CD39 drives metabolic adaption and mal-differentiation of CD4 + T cells in patients with non-small-cell lung cancer

doi: 10.1038/s41419-023-06336-4

Figure Lengend Snippet: A Healthy CD4 + T cells were pre-conditioned with A549 cells for 12 h, activated with anti-CD3/CD28 beads for 3 days, and detected for energy status. Mean ± SEM from 8 individuals in each group. B Healthy CD4 + T cells were incubated with A549 cells for 12 h and detected for the expressions of CD39, V-ATPase, and Na/K ATPase by western blot. Mean ± SEM from 4 individuals in each group. C Healthy CD4 + T cells were incubated with A549 cells for 12 h and detected for CD39 protein levels by flow cytometry. Mean ± SEM from 5 individuals in each group. D Healthy CD4 + T cells were incubated with A549 cells for 12 h and detected for CD39 mRNA expressions. Mean ± SEM from 6 individuals in each group. E , F Healthy CD4 + T cells were pre-conditioned with A549 cells in the presence or absence of CD39 inhibitor ARL67156 (10 μM) for 12 hours and activated with anti-CD3/CD28 beads for 3 days. Mean ± SEM from 6 individuals in each group. G , H Healthy CD4 + T cells were pre-conditioned with A549 cells in the presence or absence of CD39 inhibitor ARL67156 (10 μM) for 12 h, followed by activation and detection of T cell differentiation. Mean ± SEM from 6 individuals in each group. I Healthy CD4 + T cells were pre-conditioned with NSCLC patients-derived primary tumor cells in the presence or absence of CD39 inhibitor ARL67156 (10 μM) for 12 h, followed by activation and detection of T cell differentiation. Mean ± SEM from 5 individuals in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 with paired student t -test ( A – D ) and ANOVA plus Tukey method ( E – I ).

Article Snippet: Primary anti-human antibodies were used as follows: Phospho-AMPKα (Thr172) (40H9) Rabbit mAb (Cell Signaling Technology, #2535), AMPKα Antibody (Cell Signaling Technology, #2532), S6 Ribosomal Protein (54D2) Mouse mAb (Cell Signaling Technology, #2317), Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb (Cell Signaling Technology, #4858), Akt (11E7) Rabbit mAb (Cell Signaling Technology, #4685), Phospho-Akt (Ser473) (193H12) Rabbit mAb (Cell Signaling Technology, #4058), V-ATPase G1 antibody (Santa Cruz Biotechnology, sc-25333), alpha 1 Sodium Potassium ATPase/ATP1A1 antibody (Santa Cruz Biotechnology, sc-514614), and CD39 antibody (Santa Cruz Biotechnology, sc-65262).

Techniques: Incubation, Western Blot, Flow Cytometry, Activation Assay, Cell Differentiation, Derivative Assay

Journal: Cell Death & Disease

Article Title: Cancer CD39 drives metabolic adaption and mal-differentiation of CD4 + T cells in patients with non-small-cell lung cancer

doi: 10.1038/s41419-023-06336-4

Figure Lengend Snippet: A Healthy CD4 + T cells were pre-conditioned with conditioned media containing 10% and 20% A549 supernatant for 12 h, followed by activation and detection for T cell differentiation. Mean ± SEM from 5 individuals in each group. B Healthy CD4 + T cells were co-cultured with PKH67-labeled A549 cells for 12 h and detected for PKH67-labeled membrane vesicles. Mean ± SEM from 6 individuals in each group. C A549 cells were pre-treated with exosome inhibitor GW4869 (10 μM) for 24 h before pre-conditioning healthy CD4 + T cells for another 12 h. After that, CD4 + T cells were activated with anti-CD3/CD28 beads for 4 days and analyzed for T cell differentiation. Mean ± SEM from 5 individuals in each group. D CD39 protein in A549 cell-derived exosomes was detected by flow cytometry. Mean ± SEM from 6 individuals in each group. E – G Healthy CD4 + T cells were pre-treated with A549-derived exosomes for 12 h and activated with anti-CD3/CD28 beads. AMPK activation and mTOR activity were detected 3 days post-stimulation, while T-cell differentiation was determined on day 4. Mean ± SEM from 5–6 individuals in each group. H Healthy CD4 + T cells were pre-conditioned with exosomes from NSCLC patient-derived primary tumor cells for 12 h and activated with anti-CD3/CD28 beads for 4 days, and detected for T cell differentiation by flow cytometry. Mean ± SEM from 5 individuals in each group. I CD4 + T cells isolated from healthy donors, or NSCLC patients, were activated with anti-CD3/CD28 beads for 4 days, and detected for T cell differentiation by flow cytometry. Mean ± SEM from 5 individuals in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 with ANOVA plus Tukey method ( A , C ), paired student t -test ( B , D – H ) and unpaired student t -test ( I ).

Article Snippet: Primary anti-human antibodies were used as follows: Phospho-AMPKα (Thr172) (40H9) Rabbit mAb (Cell Signaling Technology, #2535), AMPKα Antibody (Cell Signaling Technology, #2532), S6 Ribosomal Protein (54D2) Mouse mAb (Cell Signaling Technology, #2317), Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb (Cell Signaling Technology, #4858), Akt (11E7) Rabbit mAb (Cell Signaling Technology, #4685), Phospho-Akt (Ser473) (193H12) Rabbit mAb (Cell Signaling Technology, #4058), V-ATPase G1 antibody (Santa Cruz Biotechnology, sc-25333), alpha 1 Sodium Potassium ATPase/ATP1A1 antibody (Santa Cruz Biotechnology, sc-514614), and CD39 antibody (Santa Cruz Biotechnology, sc-65262).

Techniques: Activation Assay, Cell Differentiation, Cell Culture, Labeling, Membrane, Derivative Assay, Flow Cytometry, Activity Assay, Isolation

Journal: Cell Death & Disease

Article Title: Cancer CD39 drives metabolic adaption and mal-differentiation of CD4 + T cells in patients with non-small-cell lung cancer

doi: 10.1038/s41419-023-06336-4

Figure Lengend Snippet: A , B A549 cells were transfected with GFP-labeled CD39 expression vector, followed by incubation with healthy CD4 + T cells for 24 h. GFP-labeled CD39 protein in interacting T cells was determined with flow cytometry and confocal microscopy. Representative and mean ± SEM from 8 independent experiments. Scale bar 5 μm. C , D Healthy CD4 + T cells of healthy donors were pre-conditioned with CD39-deficient or control A549 cells for 12 h, followed by activation and detections for metabolic adaption. Mean ± SEM from 4–6 individuals in each group. E , F Healthy CD4 + T cells were pre-conditioned with CD39-deficient or control A549 cells for 12 h, followed by activation and analyses of T cell differentiation. Mean ± SEM from 6 individuals in each group. G Healthy CD4 + T cells were pre-conditioned with exosomes derived from CD39-deficient or control A549 cells for 12 h, followed by activation and analyses of T cell differentiation. Mean ± SEM from 6 individuals in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 with paired student t -test ( A ) and ANOVA plus Tukey method ( C – G ).

Article Snippet: Primary anti-human antibodies were used as follows: Phospho-AMPKα (Thr172) (40H9) Rabbit mAb (Cell Signaling Technology, #2535), AMPKα Antibody (Cell Signaling Technology, #2532), S6 Ribosomal Protein (54D2) Mouse mAb (Cell Signaling Technology, #2317), Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb (Cell Signaling Technology, #4858), Akt (11E7) Rabbit mAb (Cell Signaling Technology, #4685), Phospho-Akt (Ser473) (193H12) Rabbit mAb (Cell Signaling Technology, #4058), V-ATPase G1 antibody (Santa Cruz Biotechnology, sc-25333), alpha 1 Sodium Potassium ATPase/ATP1A1 antibody (Santa Cruz Biotechnology, sc-514614), and CD39 antibody (Santa Cruz Biotechnology, sc-65262).

Techniques: Transfection, Labeling, Expressing, Plasmid Preparation, Incubation, Flow Cytometry, Confocal Microscopy, Control, Activation Assay, Cell Differentiation, Derivative Assay

Journal: Cell Death & Disease

Article Title: Cancer CD39 drives metabolic adaption and mal-differentiation of CD4 + T cells in patients with non-small-cell lung cancer

doi: 10.1038/s41419-023-06336-4

Figure Lengend Snippet: A PBMCs of NSCLC patients were co-cultured with corresponding PDOs or CD39 KO-PDOs for 10 days and detected for T cell differentiation. Mean ± SEM from 5 individuals in each group. B , C CD8 + T cells within the indicated PDOs were tested for productions of IFN-γ and Granzyme B by flow cytometry. Mean ± SEM from 5 individuals in each group. D CD39-deficient and control PDOs were incubated with the same patients’ PBMCs for the indicated time and analyzed for tumor outgrowth. Mean ± SEM from 5 individuals in each group. Scale bars, 200 μm. E CD39-deficient and control PDOs receiving anti-PD-1 antibody (10 μg/mL) treatment were incubated with the same patients’ PBMCs for 10 days and detected for T cell differentiation. Mean ± SEM from 5 individuals in each group. F , G PDO-infiltrating CD8 + T cells were analyzed for production of IFN-γ or Granzyme B by flow cytometry. Mean ± SEM from 5 individuals in each group. H CD39-deficient and control PDOs receiving anti-PD-1 antibody (10 μg/mL) treatment were incubated with the same patients’ PBMCs and analyzed for tumor growth at the indicated time. Mean ± SEM from 5 individuals in each group. Scale bars, 200 μm. * p < 0.05, ** p < 0.01, *** p < 0.001 with paired student t -test.

Article Snippet: Primary anti-human antibodies were used as follows: Phospho-AMPKα (Thr172) (40H9) Rabbit mAb (Cell Signaling Technology, #2535), AMPKα Antibody (Cell Signaling Technology, #2532), S6 Ribosomal Protein (54D2) Mouse mAb (Cell Signaling Technology, #2317), Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb (Cell Signaling Technology, #4858), Akt (11E7) Rabbit mAb (Cell Signaling Technology, #4685), Phospho-Akt (Ser473) (193H12) Rabbit mAb (Cell Signaling Technology, #4058), V-ATPase G1 antibody (Santa Cruz Biotechnology, sc-25333), alpha 1 Sodium Potassium ATPase/ATP1A1 antibody (Santa Cruz Biotechnology, sc-514614), and CD39 antibody (Santa Cruz Biotechnology, sc-65262).

Techniques: Cell Culture, Cell Differentiation, Flow Cytometry, Control, Incubation

Journal: PLoS ONE

Article Title: Cellular Expression Profile for Interstitial Cells of Cajal in Bladder - A Cell Often Misidentified as Myocyte or Myofibroblast

doi: 10.1371/journal.pone.0048897

Figure Lengend Snippet: Cryosections of mouse bladders were labeled with antibodies to NTPDase2 (A. green), α-SMA (B. red) and Topro-3 to label nuclei (B. blue). Color merged panels are shown on the right (C). White arrows indicate distinct NTPDase2 staining next to bladder smooth muscle cells. White scale bars = 10 µm.

Article Snippet: Affinity-purified polyclonal sheep anti-NTPDase2 antibody (AF5797) was purchased from R&D systems (Minneapolis, MN).

Techniques: Labeling, Staining

Journal: PLoS ONE

Article Title: Cellular Expression Profile for Interstitial Cells of Cajal in Bladder - A Cell Often Misidentified as Myocyte or Myofibroblast

doi: 10.1371/journal.pone.0048897

Figure Lengend Snippet: Cryosections of mouse bladders were labeled with antibodies to NTPDase2 (B. red), c-kit (A. green) and Topro-3 to label nuclei (B. blue). Color merged panels are shown on the right (C). Merged signals of NTPDase2 and c-kit are shown as yellow (C). White arrows indicate representative NTPDase2/c-kit co-localization. White scale bars = 10 µm.

Article Snippet: Affinity-purified polyclonal sheep anti-NTPDase2 antibody (AF5797) was purchased from R&D systems (Minneapolis, MN).

Techniques: Labeling

Journal: PLoS ONE

Article Title: Cellular Expression Profile for Interstitial Cells of Cajal in Bladder - A Cell Often Misidentified as Myocyte or Myofibroblast

doi: 10.1371/journal.pone.0048897

Figure Lengend Snippet: Cryosections of mouse bladders were labeled with antibodies to CD34 (A, D. green), NTPDase2 (B. red), tryptase (E. red), c-kit (G. green), and Topro-3 to label nuclei (B. E. H. blue). Color merged panels are shown on the right (C, F, I). Merged signals are shown as yellow (C. F. I). White arrows indicate representative NTPDase2/CD34 co-localization (C) or CD34/tryptase co-localization (F); White asterisks indicate CD34 positive cells with no NTPDase2 staining (C). White scale bars = 10 µm.

Article Snippet: Affinity-purified polyclonal sheep anti-NTPDase2 antibody (AF5797) was purchased from R&D systems (Minneapolis, MN).

Techniques: Labeling, Staining

Journal: PLoS ONE

Article Title: Cellular Expression Profile for Interstitial Cells of Cajal in Bladder - A Cell Often Misidentified as Myocyte or Myofibroblast

doi: 10.1371/journal.pone.0048897

Figure Lengend Snippet: Cryosections of mouse bladders were labeled with antibodies to connexin 43 (A. green), NTPDase2 (B. red) and Topro-3 to label nuclei (B. blue). Color merged panels are shown on the right (C). Merged signals of NTPDase2 and connexin 43 are shown as yellow (C). White arrows indicate representative NTPDase2/connexin 43 co-localization. White scale bars = 10 µm.

Article Snippet: Affinity-purified polyclonal sheep anti-NTPDase2 antibody (AF5797) was purchased from R&D systems (Minneapolis, MN).

Techniques: Labeling

Journal: PLoS ONE

Article Title: Cellular Expression Profile for Interstitial Cells of Cajal in Bladder - A Cell Often Misidentified as Myocyte or Myofibroblast

doi: 10.1371/journal.pone.0048897

Figure Lengend Snippet: Cryosections of mouse bladders were labeled with antibodies to vimentin (A. green), desmin (D. green), PDGFβ receptor (G. green), and NTPDase2 (B. E. H. red) and Topro-3 to label nuclei (B. blue). Color merged panels are shown on the right (C. F. I). Merged signals of NTPDase2 and vimentin, desmin, and PDGFβ receptor are shown as yellow (C. F. I). White arrows indicate representative co-localization. White asterisks indicate non-co-localized signal of smooth muscle (I) and fibroblasts (F. I). White scale bars = 10 µm.

Article Snippet: Affinity-purified polyclonal sheep anti-NTPDase2 antibody (AF5797) was purchased from R&D systems (Minneapolis, MN).

Techniques: Labeling

Journal: PLoS ONE

Article Title: Cellular Expression Profile for Interstitial Cells of Cajal in Bladder - A Cell Often Misidentified as Myocyte or Myofibroblast

doi: 10.1371/journal.pone.0048897

Figure Lengend Snippet: Cryosections of mouse bladders were labeled with antibodies to merlin/NF2 (A. green), NTPDase2 (B. red) and Topro-3 to label nuclei (B. blue). Color merged panels are shown on the right (C). Merged signals of NTPDase2 and merlin/NF2 are shown as yellow (C). White arrows indicate representative NTPDase2/merlin co-localization. White scale bars = 10 µm.

Article Snippet: Affinity-purified polyclonal sheep anti-NTPDase2 antibody (AF5797) was purchased from R&D systems (Minneapolis, MN).

Techniques: Labeling

Journal: PLoS ONE

Article Title: Cellular Expression Profile for Interstitial Cells of Cajal in Bladder - A Cell Often Misidentified as Myocyte or Myofibroblast

doi: 10.1371/journal.pone.0048897

Figure Lengend Snippet: Summary of expression profile of molecular markers in bladder ICC.

Article Snippet: Affinity-purified polyclonal sheep anti-NTPDase2 antibody (AF5797) was purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing

Journal: British Journal of Pharmacology

Article Title: Activation of P2Y 6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

doi: 10.1111/bph.12711

Figure Lengend Snippet: Primary and secondary antibodies used in immunohistochemistry experiments

Article Snippet: Observations were performed and analysed with a laser-scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code Host Dilution Supplier Primary antibodies P2Y 1 APR-009 Rabbit 1:75 Alomone P2Y 6 APR-011 Rabbit 1:100 Alomone P2Y 6 ABIN1386282 Rabbit 1:150 Antibodies Online P2X1 APR-001 Rabbit 1:50 Alomone P2X3 APR-016 Rabbit 1:100 Alomone NTPDase2 BZ1-5F Rabbit 1:400 J. Sevigny Cytokeratin 20 (CK20) M7019 Mouse 1:100 Dako Vimentin M0725 Mouse 1:150 Dako NF160 Ab7794 Mouse 1:600 Abcam VAChT AB1578 Goat 1:750 Chemicon Secondary antibodies Alexa Fluor 488 anti-rb A-21206 Donkey 1:1500 Mol.

Techniques: Immunohistochemistry

Journal: British Journal of Pharmacology

Article Title: Activation of P2Y 6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

doi: 10.1111/bph.12711

Figure Lengend Snippet: Confocal micrographs showing P2Y6, P2Y1 and NTPDase2 immunoreactivity in transverse sections of the detrusor smooth layer of rat urinary bladder. To facilitate visualization of small cholinergic nerve terminals staining for VAChT (red) images correspond to the intensity projections over Z axis of five to six confocal microscopy stacks taken at the smooth muscle layer. No co-localization was found between P2Y6 receptor (green) and VAChT (red) immunoreactivity. Conversely, VAChT-positive cholinergic nerve terminals (red) stained positively with antibodies against the P2Y1 receptor and E-NTPDase2 (green); yellow staining denotes co-localization. Nucleic DNA is stained with DAPI (blue). Scale bars = 50 μm.

Article Snippet: Observations were performed and analysed with a laser-scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code Host Dilution Supplier Primary antibodies P2Y 1 APR-009 Rabbit 1:75 Alomone P2Y 6 APR-011 Rabbit 1:100 Alomone P2Y 6 ABIN1386282 Rabbit 1:150 Antibodies Online P2X1 APR-001 Rabbit 1:50 Alomone P2X3 APR-016 Rabbit 1:100 Alomone NTPDase2 BZ1-5F Rabbit 1:400 J. Sevigny Cytokeratin 20 (CK20) M7019 Mouse 1:100 Dako Vimentin M0725 Mouse 1:150 Dako NF160 Ab7794 Mouse 1:600 Abcam VAChT AB1578 Goat 1:750 Chemicon Secondary antibodies Alexa Fluor 488 anti-rb A-21206 Donkey 1:1500 Mol.

Techniques: Staining, Confocal Microscopy

Journal: British Journal of Pharmacology

Article Title: Activation of P2Y 6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

doi: 10.1111/bph.12711

Figure Lengend Snippet: Schematic representation of the putative mechanisms underlying the control of the voiding frequency by urothelial UDP-sensitive P2Y6 receptors in the anaesthetized rat. Activation of P2Y6 receptors on distended umbrella cells during bladder filling increased, by threefold, the release of ATP from the urothelium. Released ATP, acting via multiple urothelial P2 purinoceptors, triggers a self-regenerating purinergic wave propagating to sensory nerve afferents endowed with P2X3 receptors to initiate the voiding reflex. Bladder activity may be partly reversed by the hydrolysis of ATP into ADP by E-NTPDases, namely E-NTPDase2 located in the lamina propria (probably on interstitial cells) and on cholinergic nerve efferents. ADP accumulation at the neuromuscular synapse decreases ACh release and smooth muscle contraction through the activation of prejunctional inhibitory P2Y1 receptors. The diagram also shows the locus of action of the main drugs used in this study.

Article Snippet: Observations were performed and analysed with a laser-scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code Host Dilution Supplier Primary antibodies P2Y 1 APR-009 Rabbit 1:75 Alomone P2Y 6 APR-011 Rabbit 1:100 Alomone P2Y 6 ABIN1386282 Rabbit 1:150 Antibodies Online P2X1 APR-001 Rabbit 1:50 Alomone P2X3 APR-016 Rabbit 1:100 Alomone NTPDase2 BZ1-5F Rabbit 1:400 J. Sevigny Cytokeratin 20 (CK20) M7019 Mouse 1:100 Dako Vimentin M0725 Mouse 1:150 Dako NF160 Ab7794 Mouse 1:600 Abcam VAChT AB1578 Goat 1:750 Chemicon Secondary antibodies Alexa Fluor 488 anti-rb A-21206 Donkey 1:1500 Mol.

Techniques: Activation Assay, Activity Assay