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: On each plate, a standard curve of recombinant human CD73 protein (R&D Systems, 5795-EN) was run.

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: On each plate, a standard curve of recombinant human CD73 protein (R&D Systems, 5795-EN) was run.

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 3. CD73 on tumor cells and myeloid cells promotes mouse PDAC. (A) CD73-positive (pos) or -negative (neg) KPC tumor cells were injected s.c. into C57BL/6 mice and treated with gemcitabine (gem; i.p. 100 mg/kg) on days 5 and 8. Mean tumor sizes are shown ± SEM (n=9–11). (B) KPC tumors were analyzed at day 11 by qPCR for expression of selected immune genes. Data represent mean relative expression ± SEM compared to CD73-positive tumors. (C) KPC tumors were analyzed at day 11 by FACS to assess the proportion of CD11b+Ly6G−Ly6Clo/− cells (TAMs) expressing CD206. Data represent individual and mean frequencies of TAM ± SEM gated on CD45+ cells. (D-E) CD73-negative KPC tumor cells were injected s.c. into CD73fl/fl LysMCre−/− mice (n=9) and into CD73fl/fl LysMCre+/− mice (n=12). On day 11, tumors were weighed (D) and the proportion of CD206+ TAMs was assessed by FACS. Data are representative of 2 independent experiments (n=9–10/group). Statistical comparisons were performed using one-way ANOVA comparing indicated groups (A, C), multiple t-test or unpaired t-test (B, D, E). *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

Article Snippet: On each plate, a standard curve of recombinant human CD73 protein (R&D Systems, 5795-EN) was run.

Techniques: Injection, 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 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: On each plate, a standard curve of recombinant human CD73 protein (R&D Systems, 5795-EN) was run.

Techniques: Injection

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 5. CD73 promotes DNA damage repair and suppresses cGAS-STING activation. (A-B) CD73-positive (pos) or -negative (neg) KPC and PANC1 cells were exposed to increasing concentrations of gemcitabine, and proliferation was measured using CellTiter-Glo (Promega) after 48 hours of treatment. Data represents the average relative luminescence units (RLU) ± SEM of triplicates and is representative of 4 independent experiments. (C) Meta-analysis of CD73 gene association with sensitivity to gemcitabine in PDAC cell lines from the Genentech Cell Line Screening Initiative (gCSI), the Genomics of Drug Sensitivity in Cancer (GDSC), and the Cancer Therapeutics Response Portal (CTRP) datasets. Forest plot displaying the C-Index and 95% confidence intervals (CI) for each dataset. Horizontal bars represent the 95% confidence intervals of effect-size. The blue diamond represents the overall effect of the variable. (D) Representative images of γ-H2AX foci detected by immunofluorescence. (E) Number of γ-H2AX foci detected by immunofluorescence in CD73-positive or -negative KPC tumor cells following 48 hours of gemcitabine treatment at the indicated doses (n=4; mean ± SEM). (F) Number of γ-H2AX foci over time detected by immunofluorescence in KPC tumor cells following 1 Gy irradiation (n=3; mean ± SEM). (G) Number of γ-H2AX foci detected in human PANC1 cells treated with gemcitabine, CD73 inhibitor AB680, and/or A2B receptor agonist BAY 60–6583 (n=3; mean ± SEM). (H) Number of γ-H2AX foci detection after 48 hours of gemcitabine treatment in KPC tumor cells, in presence or absence of BAY 60–6583 (A2B receptor agonist)(n=3; mean ± SEM). (I) CD73-positive or -negative KPC tumor cells were exposed to gemcitabine (20 nM) for 48 hours and cGAMP was measured in supernatants by ELISA (n=3; mean ± SEM). (J) Following gemcitabine exposition, cGAS- induced gene expression in KPC cells was measured by qPCR (n=2; mean ± SEM). (K) CD73-positive KPC tumor cells expressing cGAS or not were injected in mice (n=10 per group). Some groups were treated with AB680 (peri-tumoral, 10mg/kg daily from day 6 to 11). Means ± SEM tumor growth at day 20 are shown (experiment performed once). Statistical significance was determined with Student T test (A-B, E-K). *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

Article Snippet: On each plate, a standard curve of recombinant human CD73 protein (R&D Systems, 5795-EN) was run.

Techniques: Activation Assay, Immunofluorescence, Irradiation, Enzyme-linked Immunosorbent Assay, Gene Expression, Expressing, Injection

Journal: Neural Regeneration Research

Article Title: MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

doi: 10.4103/NRR.NRR-D-23-01064

Figure Lengend Snippet: Summary of antibody dilutions and conditions used in western blot analysis

Article Snippet: PSN1 rabbit polyclonal , 1:1000 , 16163-1-AP , Proteintech, Rosemont, IL, USA.

Techniques: Western Blot

Journal: Neural Regeneration Research

Article Title: MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

doi: 10.4103/NRR.NRR-D-23-01064

Figure Lengend Snippet: Modulation of AD proteins by miR-502-3p. (A) Immunoblot analysis of amyloid precursor protein (APP), tubulin associated unit (Tau), presenilin-1 (PSN1) and presenilin-2 (PSN2) and -actin protein in hippocampal neurons (HT22) cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. The levels of AD proteins were changed in miR-502-3p agomiR and antagomiR transfected cells. (B) Densitometry analysis of APP, Tau, PSN1 and PSN2 blot shows increased protein levels in miR-502-3p agomiR transfected cells compared with scramble control, while the cells treated with miR-502-3p antagomiR shows a reduced level of all AD proteins compared with agomiRs. For statistical analysis, Student’s t -test was used for analyzing the data between three groups of samples such as scramble control, agomiRs, and antagomiRs. Experiments were performed in triplicates. The P -values < 0.05 were considered statistically significant. AD: Alzheimer’s disease; miR: microRNA.

Article Snippet: PSN1 rabbit polyclonal , 1:1000 , 16163-1-AP , Proteintech, Rosemont, IL, USA.

Techniques: Western Blot, Transfection, Control

Journal: Scientific Reports

Article Title: Role of P2X 4 Receptor in Mouse Voiding Function

doi: 10.1038/s41598-018-20216-4

Figure Lengend Snippet: Immunoprecipitation with anti-P2X 1 antibody. Antibodies to P2X 1 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads were resolved by SDS-PAGE, and Western blots were probed with A) P2X 1, B) P2X 4 or C) Nt5e antibodies. ( A ) Left and right panels show P2X 1 immunoblots on IP and FT lysates from wild type and P2X 4 −/− mice. Monomeric P2X 1 can be seen highly concentrated in the pulldown fraction at 50 kDa. Little P2X 1 appears in FT. ( B ) P2X 4 antibody detects P2X 4 as a band at 70 kDa in wild type, but is completely absent in P2X 4 −/− mice. The antibody shows minor cross-reactivity to possibly P2X 1 . Note, there is no evidence of the 70 kDa band in the IP lane. ( C ) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X 1 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

Article Snippet: The isolated proteins were resolved on 8–16% polyacrylamide gradient gels under reducing condition (+0.1 M DTT and 95 °C for 5 min before loading), and further blotted and probed with anti-P2X 1 , anti-P2X 4 and anti-5′-nucleotidase (NT5E) (Catalogue #: MAB44881, R&D system) antibodies for protein detection.

Techniques: Immunoprecipitation, Incubation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Protein Binding

Journal: Scientific Reports

Article Title: Role of P2X 4 Receptor in Mouse Voiding Function

doi: 10.1038/s41598-018-20216-4

Figure Lengend Snippet: Immunoprecipitation with anti-P2X 4 antibody. Antibodies to P2X 4 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads, were resolved by SDS-PAGE, and Western blots were probed with A ) P2X 1, B) P2X 4 or C ) Nt5e antibodies. ( A ) Left and right panels show P2X 1 immunoblots on IP and FT lysates from wild type and P2X 4 −/− mice. P2X 1 is highly concentrated in the FT fractions. Minor potential P2X 1 staining appears in the IP lane, however this is due to P2X 4 antibody cross-reacting and pulling down some P2X 1 . ( B ) P2X 4 antibody detects P2X 4 as a band at 70 kDa in wild type IP lane, but is absent in P2X 4 −/− mice. The antibody shows minor cross-reactivity to P2X 1 (50 kDa band). ( C ) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X 4 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

Article Snippet: The isolated proteins were resolved on 8–16% polyacrylamide gradient gels under reducing condition (+0.1 M DTT and 95 °C for 5 min before loading), and further blotted and probed with anti-P2X 1 , anti-P2X 4 and anti-5′-nucleotidase (NT5E) (Catalogue #: MAB44881, R&D system) antibodies for protein detection.

Techniques: Immunoprecipitation, Incubation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Staining, Protein Binding

Journal: Purinergic Signalling

Article Title: Characterization of the N 6 -etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

doi: 10.1007/s11302-020-09699-x

Figure Lengend Snippet: When N6-etheno-ATP (a) and N6-etheno-AMP (b) were incubated for 30 min at 30 °C in the absence of ecto-nucleotidases, the only chromatographic peaks observed were intact N6-etheno-ATP and intact N6-etheno-AMP, respectively, thus indicating that N6-etheno-ATP and N6-etheno-AMP were chemically stable under these test conditions. When N6-etheno-ATP was incubated for 30 min at 30 °C in the presence of either 20 ng of rhCD39 (c), 80 ng of rhENPP-1 (d), 40 ng of rhENTPD2 (e), or 11 ng of rhENTPD3 (f), N6-etheno-ATP was essentially quantitatively converted to N6-etheno-AMP. When N6-etheno-AMP (g) was incubated for 30 min at 30 °C in the presence of rhCD73 (40 ng), all of the N6-etheno-AMP was recovered as N6-etheno-adenosine (ADO)

Article Snippet: Recombinant human CD39 (rhCD39), recombinant human CD73 (rhCD73), recombinant human ecto-nucleotide pyrophosphatase/phosphodiesterase family member 1 (rhENPP-1), recombinant human ectonucleoside triphosphate diphosphohydrolase family member 2 (rhENTPD2), and recombinant human ectonucleoside triphosphate diphosphohydrolase family member 3 (rhENTPD3) were obtained from R&D Systems (Minneapolis, MN; catalog numbers 4397-EN-010, 5795-EN-010, 6136-EN-010, 6087-EN-010, and 4400-EN-010, respectively).

Techniques: Incubation

Journal: Purinergic Signalling

Article Title: Characterization of the N 6 -etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

doi: 10.1007/s11302-020-09699-x

Figure Lengend Snippet: Scatter plots show the percentage (%) of applied substrate (either the natural adenine nucleotide substrate or the corresponding etheno-bridged adenine nucleotide substrate, both at 1 μmol/L) that remained or was recovered as product (either the natural product or corresponding etheno-bridged product) after incubation (5 min at 30 °C) with recombinant human (rh) ecto-nucleotidases a rhENPP-1, b rhENTPD2, c rhENTPD3, d rhCD73, or e rhCD39. For each ecto-nucleotidase, the amount of enzyme incubated with substrate was selected to only partially metabolize the natural adenine nucleotide substrate. eATP = N6-etheno-ATP; eADP = N6-etheno-ADP; eAMP = N6-etheno-AMP; eADO = N6-etheno-adenosine (eADO). *P < 0.05 versus corresponding natural substrate. All individual data points are provided along with the means and SDs

Article Snippet: Recombinant human CD39 (rhCD39), recombinant human CD73 (rhCD73), recombinant human ecto-nucleotide pyrophosphatase/phosphodiesterase family member 1 (rhENPP-1), recombinant human ectonucleoside triphosphate diphosphohydrolase family member 2 (rhENTPD2), and recombinant human ectonucleoside triphosphate diphosphohydrolase family member 3 (rhENTPD3) were obtained from R&D Systems (Minneapolis, MN; catalog numbers 4397-EN-010, 5795-EN-010, 6136-EN-010, 6087-EN-010, and 4400-EN-010, respectively).

Techniques: Incubation, Recombinant

Journal: Purinergic Signalling

Article Title: Characterization of the N 6 -etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

doi: 10.1007/s11302-020-09699-x

Figure Lengend Snippet: To determine initial reaction velocities, CD39 (10 ng) was incubated with high concentrations of substrates (25 to 200 μmol/L) for 10 min at 30 °C. In panel a, substrates were either ATP or N6-etheno-ATP and the downstream products (ADP + AMP or N6-etheno-ADP + N6-etheno-AMP) were measured. In panel b, substrates were either ADP or N6-etheno-ADP and the downstream products (AMP or N6-etheno-AMP) were measured. The experiment in panel c was similar to that described for panel b with the exception that the substrates were AMP or N6-etheno-AMP, the enzyme was CD73 (0.25 ng), and the measured products were adenosine and N6-etheno-ADO. eATP = N6-etheno-ATP; eADP = N6-etheno-ADP; eAMP = N6-etheno-AMP. Values represent means ± SDs

Article Snippet: Recombinant human CD39 (rhCD39), recombinant human CD73 (rhCD73), recombinant human ecto-nucleotide pyrophosphatase/phosphodiesterase family member 1 (rhENPP-1), recombinant human ectonucleoside triphosphate diphosphohydrolase family member 2 (rhENTPD2), and recombinant human ectonucleoside triphosphate diphosphohydrolase family member 3 (rhENTPD3) were obtained from R&D Systems (Minneapolis, MN; catalog numbers 4397-EN-010, 5795-EN-010, 6136-EN-010, 6087-EN-010, and 4400-EN-010, respectively).

Techniques: Incubation