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AMS Biotechnology murine cd73
Murine Cd73, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/murine+cd73/Biotinylated+Human+CD73+%2F+NT5E+Protein%2C+His%2CAvitag/pm38206570-217-11-13
Average 96 stars, based on 1 article reviews

murine cd73 - by Bioz Stars, 2026-09

96/100 stars

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Article Title: Efficacy and pharmacodynamic effect of anti-CD73 and anti-PD-L1 monoclonal antibodies in combination with cytotoxic therapy: observations from mouse tumor models
Article Snippet: A 96 half well plate (Corning, 3690) was coated with streptavidin (Invitrogen 434,301) at 2ug/ml overnight with no shaking, washed, and then blocked for one hour using 1% BSA (Sigma, A6003) in PBS. .. The plate was washed, and 0.5ug/ml biotinylated human (Amsbio, AMS.CD3-H82E3) or murine CD73 (Amsbio, AMS.79416–1) was incubated for one hour. .. Twelve point serial dilutions of oleclumab and aCD73 mIgG1 clone 10.3 (In house, 864–0.014632 ng/ml) were added and incubated for two hours, before incubation of 1:2500 HRP labeled anti mouse Fc antibody (Sigma, A2554), or 1:5000 HRP labeled anti-human Fc antibody (Sigma, A0170) for one hour.

Article Title: Efficacy and pharmacodynamic effect of anti-CD73 and anti-PD-L1 monoclonal antibodies in combination with cytotoxic therapy: observations from mouse tumor models.
Article Snippet: A 96 half well plate (Corning, 3690) was coated with streptavidin (Invitrogen 434,301) at 2ug/ml overnight with no shaking, washed, and then blocked for one hour using 1% BSA (Sigma, A6003) in PBS. .. The plate was washed, and 0.5ug/ml biotinylated human (Amsbio, AMS.CD3-H82E3) or murine CD73 (Amsbio, AMS.79416–1) was incubated for one hour. .. Twelve point serial dilutions of oleclumab and aCD73 mIgG1 clone 10.3 (In house, 864–0.014632 ng/ml) were added and incubated for two hours, before incubation of 1:2500 HRP labeled anti mouse Fc antibody (Sigma, A2554), or 1:5000 HRP labeled anti-human Fc antibody (Sigma, A0170) for one hour.

Incubation:

Article Title: Efficacy and pharmacodynamic effect of anti-CD73 and anti-PD-L1 monoclonal antibodies in combination with cytotoxic therapy: observations from mouse tumor models.
Article Snippet: A 96 half well plate (Corning, 3690) was coated with streptavidin (Invitrogen 434,301) at 2ug/ml overnight with no shaking, washed, and then blocked for one hour using 1% BSA (Sigma, A6003) in PBS. .. The plate was washed, and 0.5ug/ml biotinylated human (Amsbio, AMS.CD3-H82E3) or murine CD73 (Amsbio, AMS.79416–1) was incubated for one hour. .. Twelve point serial dilutions of oleclumab and aCD73 mIgG1 clone 10.3 (In house, 864–0.014632 ng/ml) were added and incubated for two hours, before incubation of 1:2500 HRP labeled anti mouse Fc antibody (Sigma, A2554), or 1:5000 HRP labeled anti-human Fc antibody (Sigma, A0170) for one hour.

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bsAb AD2×1E9 binds simultaneously with both Fab arms, is resistant to APCP-mediated epitope sequestration and exhibits increased Ab deposition. (A) Competition of fluorescently labeled Abs (AD2-AF488, 1E9-AF488, MEDI9447-AF594, bsAb AD2×1E9-AF488, 5 µg/mL) with preincubated non-labeled Abs (indicated to the left, 10 µg/mL) and analyzed by flow cytometry. Peaks shifted to the left indicate blocking of the labeled Ab. Panel (A) is representative of three independent experiments. (B, C) Increased binding by the AD2-AF488 in the presence of 1E9 and bsAb 1E9xb12 was observed, whereas binding of 1E9-AF488 was not affected by the presence of AD2 or bsAb AD2xb12, as determined by flow cytometry analysis. (D) Ab binding in the presence or absence of APCP as determined by flow cytometry analysis. Epitope recognition by anti-CD73 Abs was detected by addition of a secondary fluorescent-labeled Ab. Mean±SEM from three independent experiments is shown. Statistical difference was determined using paired t-tests. (E) Schematic representation of the epitope interactions. (F) Flow cytometry analysis of Ab binding to MDA-MB-231 cells using a similar setup as in panel (D). Mean±SEM from three independent experiments is shown. Statistical difference was determined by Student’s t-test (D) or two-way ANOVA (F) method followed by Bonferoni’s multiple comparison testing. *0.05>p≥0.01; **0.01>p≥0.001; ***0.001>p. Abs, antibodies; ANOVA, analysis of variance; bsAbs, bispecific antibodies; APCP, adenosine 5′-α,β-(methylene)diphosphate; NS non-significant.

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting two distinct epitopes on human CD73 with a bispecific antibody improves anticancer activity

doi: 10.1136/jitc-2022-004554

Figure Lengend Snippet: bsAb AD2×1E9 binds simultaneously with both Fab arms, is resistant to APCP-mediated epitope sequestration and exhibits increased Ab deposition. (A) Competition of fluorescently labeled Abs (AD2-AF488, 1E9-AF488, MEDI9447-AF594, bsAb AD2×1E9-AF488, 5 µg/mL) with preincubated non-labeled Abs (indicated to the left, 10 µg/mL) and analyzed by flow cytometry. Peaks shifted to the left indicate blocking of the labeled Ab. Panel (A) is representative of three independent experiments. (B, C) Increased binding by the AD2-AF488 in the presence of 1E9 and bsAb 1E9xb12 was observed, whereas binding of 1E9-AF488 was not affected by the presence of AD2 or bsAb AD2xb12, as determined by flow cytometry analysis. (D) Ab binding in the presence or absence of APCP as determined by flow cytometry analysis. Epitope recognition by anti-CD73 Abs was detected by addition of a secondary fluorescent-labeled Ab. Mean±SEM from three independent experiments is shown. Statistical difference was determined using paired t-tests. (E) Schematic representation of the epitope interactions. (F) Flow cytometry analysis of Ab binding to MDA-MB-231 cells using a similar setup as in panel (D). Mean±SEM from three independent experiments is shown. Statistical difference was determined by Student’s t-test (D) or two-way ANOVA (F) method followed by Bonferoni’s multiple comparison testing. *0.05>p≥0.01; **0.01>p≥0.001; ***0.001>p. Abs, antibodies; ANOVA, analysis of variance; bsAbs, bispecific antibodies; APCP, adenosine 5′-α,β-(methylene)diphosphate; NS non-significant.

Article Snippet: A sequence encoding a chimeric CD73 molecule comprising amino acid number 27-549 of human CD73 flanked by 1-28 and 551-579 murine CD73 was inserted into the pLenti6.2-ccdB-3xFLAG-V5 plasmid using the EcoRV and SacII restriction sites. pLenti6.2-ccdB-3xFLAG-V5 was a gift from Susan Lindquist & Mikko Taipale (Addgene plasmid # 87071; http://n2t.net/addgene :87071; RRID:Addgene_87071).

Techniques: Labeling, Flow Cytometry, Blocking Assay, Binding Assay, Comparison

$bsAb AD2×1E9 induces potent Fc-mediated cell death leading to anticancer activity. (A) Merged bright field and immunofluorescence images showing the surface pattern of CD73 on MDA-MB-231 cells following incubation with the indicated Abs, showing distinct clusters when incubated with AD2-derived Abs. (B) Cancer cell viability analysis (luminescence) following incubation with Abs (10 µg/mL) and human PBMCs at an effector-target (ET) ratio of 100:1 after 72 hours, demonstrating epitope-specific cancer cell killing. A Tukey box comprising data from peripheral blood mononuclear cells (PBMCs) of 24 healthy donors is shown. Statistical difference was determined by ANOVA followed by Bonferoni’s multiple comparison testing. (C) Similar setup as for (B) but using purified Natural killer (NK) cells instead of PBMCs at an ET ratio of 12.5:1. A representative of two experiments is shown. (D) Similar setup as for (B, C) but comparing PBMCs, NK cells and PBMCs depleted of NK cells. The added NK to cancer cell ratio is 14 for both PBMCs and NK cell-enriched fractions, and five for NK cell-depleted fractions. A representative of three independent experiments is shown. (E) Similar setup as for (B–D) but comparing the effect of enriched monocytes and T cells separately and in combination. Monocytes and T cells were added at an ET ratio of 10 and 50, respectively. A representative of four experiments is shown. Statistical difference in (D–E) was determined by Student’s t-tests. (F) Female age-matched NOG mice were transplanted with human PBMCs (2×10 7 ) from healthy donors, two mice per donor, each assigned to an intravenous injection with MDA-MB-231-luc2+ cells (10 5 ) preincubated with either bsAb AD2×1E9 or b12 Ab (n=5). Mean±SEM is shown. Statistical difference was determined by two-way ANOVA followed by Bonferoni’s multiple comparison testing. (G) Similar to (F) showing raw in vivo bioimaging data demonstrating reduced cancer growth across PBMC donor matched mice. (H) As in (F) with a total of 10 donors (AD2 (n=10), 1E9 (n=10) and bsAb AD2×1E9 (n=15) and b12(n=15)) with read-out after 11 days. A Tukey box plot is shown, showing similar activity ranks of the anti-CD73 Abs as in (B) and . Statistical difference was determined by Student’s t-test. (I) Similar setup as for (H) but evaluating the effect of Fc interactions, T cells and monocytes, respectively. Quantification is based on immunohistochemistry as described in the Materials and methods section. Statistical difference was determined by the Mann-Whitney test. *0.05>p≥0.01; **0.01>p≥0.001; ***0.001>p. Abs, antibodies; ANOVA, analysis of variance; bsAbs, bispecific antibodies;; NS non-significant.$

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting two distinct epitopes on human CD73 with a bispecific antibody improves anticancer activity

doi: 10.1136/jitc-2022-004554

Figure Lengend Snippet: bsAb AD2×1E9 induces potent Fc-mediated cell death leading to anticancer activity. (A) Merged bright field and immunofluorescence images showing the surface pattern of CD73 on MDA-MB-231 cells following incubation with the indicated Abs, showing distinct clusters when incubated with AD2-derived Abs. (B) Cancer cell viability analysis (luminescence) following incubation with Abs (10 µg/mL) and human PBMCs at an effector-target (ET) ratio of 100:1 after 72 hours, demonstrating epitope-specific cancer cell killing. A Tukey box comprising data from peripheral blood mononuclear cells (PBMCs) of 24 healthy donors is shown. Statistical difference was determined by ANOVA followed by Bonferoni’s multiple comparison testing. (C) Similar setup as for (B) but using purified Natural killer (NK) cells instead of PBMCs at an ET ratio of 12.5:1. A representative of two experiments is shown. (D) Similar setup as for (B, C) but comparing PBMCs, NK cells and PBMCs depleted of NK cells. The added NK to cancer cell ratio is 14 for both PBMCs and NK cell-enriched fractions, and five for NK cell-depleted fractions. A representative of three independent experiments is shown. (E) Similar setup as for (B–D) but comparing the effect of enriched monocytes and T cells separately and in combination. Monocytes and T cells were added at an ET ratio of 10 and 50, respectively. A representative of four experiments is shown. Statistical difference in (D–E) was determined by Student’s t-tests. (F) Female age-matched NOG mice were transplanted with human PBMCs (2×10 7 ) from healthy donors, two mice per donor, each assigned to an intravenous injection with MDA-MB-231-luc2+ cells (10 5 ) preincubated with either bsAb AD2×1E9 or b12 Ab (n=5). Mean±SEM is shown. Statistical difference was determined by two-way ANOVA followed by Bonferoni’s multiple comparison testing. (G) Similar to (F) showing raw in vivo bioimaging data demonstrating reduced cancer growth across PBMC donor matched mice. (H) As in (F) with a total of 10 donors (AD2 (n=10), 1E9 (n=10) and bsAb AD2×1E9 (n=15) and b12(n=15)) with read-out after 11 days. A Tukey box plot is shown, showing similar activity ranks of the anti-CD73 Abs as in (B) and . Statistical difference was determined by Student’s t-test. (I) Similar setup as for (H) but evaluating the effect of Fc interactions, T cells and monocytes, respectively. Quantification is based on immunohistochemistry as described in the Materials and methods section. Statistical difference was determined by the Mann-Whitney test. *0.05>p≥0.01; **0.01>p≥0.001; ***0.001>p. Abs, antibodies; ANOVA, analysis of variance; bsAbs, bispecific antibodies;; NS non-significant.

Techniques: Activity Assay, Immunofluorescence, Incubation, Derivative Assay, Comparison, Purification, Injection, In Vivo, Immunohistochemistry, MANN-WHITNEY

bsAb AD2×1E9 displays enhanced potency and efficacy in terms of inhibiting the enzymatic activity of CD73. (A) Enzymatic activity of CD73 on intact cells (MDA-MB-231) on incubation with serially diluted Ab solutions as measured by radiometry. Means±SEM from three to four independent experiments are shown. (B) Comparison of the enzymatic inhibition in panel (A) at 30 µg/mL. Mean±SD is shown. Statistical difference was determined by one-way ANOVA method followed by Sidak’s multiple comparison testing. (C) Enzymatic activity of CD73 using membrane preparations from MDA-MB-231 cells on incubation with serially diluted Ab solutions as measured by radiometry. Mean±SEM from three to four independent experiments is shown. (D) Comparison of the enzymatic activity shown in panels (A) and (C) at concentrations that induced the largest effect, demonstrating similar activity of CD73 using intact cells and purified membranes. Mean±SD is shown. (E) Comparison of the enzymatic inhibition of recombinant soluble human CD73 on incubation with serially diluted Ab as measured by radiometry. Means±SEM from two independent experiments are shown. Statistical difference was determined by two-way ANOVA method followed by Sidak’s multiple comparison testing. *0.05>p≥0.01; **0.01>p≥0.001; ***0.001>p. Abs, antibodies; ANOVA, analysis of variance; bsAb, bispecific antibodies; NS non-significant.

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting two distinct epitopes on human CD73 with a bispecific antibody improves anticancer activity

doi: 10.1136/jitc-2022-004554

Figure Lengend Snippet: bsAb AD2×1E9 displays enhanced potency and efficacy in terms of inhibiting the enzymatic activity of CD73. (A) Enzymatic activity of CD73 on intact cells (MDA-MB-231) on incubation with serially diluted Ab solutions as measured by radiometry. Means±SEM from three to four independent experiments are shown. (B) Comparison of the enzymatic inhibition in panel (A) at 30 µg/mL. Mean±SD is shown. Statistical difference was determined by one-way ANOVA method followed by Sidak’s multiple comparison testing. (C) Enzymatic activity of CD73 using membrane preparations from MDA-MB-231 cells on incubation with serially diluted Ab solutions as measured by radiometry. Mean±SEM from three to four independent experiments is shown. (D) Comparison of the enzymatic activity shown in panels (A) and (C) at concentrations that induced the largest effect, demonstrating similar activity of CD73 using intact cells and purified membranes. Mean±SD is shown. (E) Comparison of the enzymatic inhibition of recombinant soluble human CD73 on incubation with serially diluted Ab as measured by radiometry. Means±SEM from two independent experiments are shown. Statistical difference was determined by two-way ANOVA method followed by Sidak’s multiple comparison testing. *0.05>p≥0.01; **0.01>p≥0.001; ***0.001>p. Abs, antibodies; ANOVA, analysis of variance; bsAb, bispecific antibodies; NS non-significant.

Techniques: Activity Assay, Incubation, Comparison, Inhibition, Membrane, Purification, Recombinant

Targeting CD73 with the bsAb AD2×1E9 inhibits tumor growth. (A) Schematic outline of the chimeric mouse/human CD73 construct. Numbers indicate amino acid residues. Numbers in parentheses indicate their location in syngeneic species. (B, C) Flow cytometry analysis demonstrating lack of surface mouse CD73 on CT26.CL25 cells (B) and expression of human CD73 on CT26.CL25-hCD73 cells (C). Representative of two independent experiments is shown. (D) Enzymatic activity of chimeric CD73 on incubation with 10 µg/mL Ab as measured by the Malachite green assay, illustrating inhibitory activity of the bsAb AD2×1E9. A representative of three independent experiments performed in triplicates is shown. (E) Flow cytometry analysis illustrating removal of surface CD73 by the AD2 Ab on 3-hour incubation. (F) Schematic illustration of the ACT experiment workflow. (G) Female NOG mice were injected subcutaneously with 7×10 5 CT26.CL25-hCD73 cells. On day 1, mice were injected intravenously with PBS or 2×10 7 splenocytes from female Balb/c CT26.CL25-hCD73 rejecting mice. On days 1 and 8, mice were treated with 200 µg Fc-silenced Ab or PBS intraperitoneally. Mean±SEM is shown. n=14 (AD2×1E9), n=7 (AD2), n=7 (1E9), n=7 (b12). (H) Mice were challenged as described in (G) and left untreated (n=7), treated with AD2×1E9 as monotherapy (n=7), or with AD2×1E9 in combination with either 2×10 7 splenocytes from unvaccinated mice (n=6), 2×10 7 splenocytes from vaccinated mice (n=6), or 5×10 6 T cells from vaccinated mice (n=4). All bsAb constructs were Fc-silenced. Statistical difference in survival was determined by log-rank (Mantel-Cox) test. *p<0.05; **0.01>p≥0.001; ***p<0.001. Abs, antibodies; bsAbs, bispecific antibodies; ACT, adoptive cell transfer.

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting two distinct epitopes on human CD73 with a bispecific antibody improves anticancer activity

doi: 10.1136/jitc-2022-004554

Figure Lengend Snippet: Targeting CD73 with the bsAb AD2×1E9 inhibits tumor growth. (A) Schematic outline of the chimeric mouse/human CD73 construct. Numbers indicate amino acid residues. Numbers in parentheses indicate their location in syngeneic species. (B, C) Flow cytometry analysis demonstrating lack of surface mouse CD73 on CT26.CL25 cells (B) and expression of human CD73 on CT26.CL25-hCD73 cells (C). Representative of two independent experiments is shown. (D) Enzymatic activity of chimeric CD73 on incubation with 10 µg/mL Ab as measured by the Malachite green assay, illustrating inhibitory activity of the bsAb AD2×1E9. A representative of three independent experiments performed in triplicates is shown. (E) Flow cytometry analysis illustrating removal of surface CD73 by the AD2 Ab on 3-hour incubation. (F) Schematic illustration of the ACT experiment workflow. (G) Female NOG mice were injected subcutaneously with 7×10 5 CT26.CL25-hCD73 cells. On day 1, mice were injected intravenously with PBS or 2×10 7 splenocytes from female Balb/c CT26.CL25-hCD73 rejecting mice. On days 1 and 8, mice were treated with 200 µg Fc-silenced Ab or PBS intraperitoneally. Mean±SEM is shown. n=14 (AD2×1E9), n=7 (AD2), n=7 (1E9), n=7 (b12). (H) Mice were challenged as described in (G) and left untreated (n=7), treated with AD2×1E9 as monotherapy (n=7), or with AD2×1E9 in combination with either 2×10 7 splenocytes from unvaccinated mice (n=6), 2×10 7 splenocytes from vaccinated mice (n=6), or 5×10 6 T cells from vaccinated mice (n=4). All bsAb constructs were Fc-silenced. Statistical difference in survival was determined by log-rank (Mantel-Cox) test. *p<0.05; **0.01>p≥0.001; ***p<0.001. Abs, antibodies; bsAbs, bispecific antibodies; ACT, adoptive cell transfer.

Techniques: Construct, Flow Cytometry, Expressing, Activity Assay, Incubation, Malachite Green Assay, Injection

(A) Representative image of Passage 3 mesenchymal stem cells (MSCs), 400× magnification. (B) Flow cytometry represents BM-MSC characterization. Cells were labeled with anti–CD73-FITC, anti–CD29-PE, and anti–CD90-PerCP-Cy5.5. (C) Induced differentiation of BM-MSCs was performed by culturing the cells in maintenance media or differentiation media (osteogenic or adipogenic). After week 3, cells in the osteocytic media were counter stained with Von Kossa to assess calcium deposit. Representative images are shown for osteocytes (top) and adipocyte (bottom). (D) Whole-cell lysates from BM-MSCs or BM stromal cells were immunoprecipitated with anti-CDH2 or IgG (Antibody control) and then electrophorese on 12% SDS–PAGE. The proteins were transferred onto PVDF membrane for blotting with anti-Cx43. (E) Real time PCR for CDH2. The results were normalized for β-actin and then presented as the mean ± SD (n = 5) relative expression. (F) Western blot with extracts from BC cell subsets: Oct4a-GFP hi, med or lo . (G, H) Top panel: Western blot for CDH2 (F) and Cx43 (G) using whole-cell extracts from MDA-MB-231, transfected with shRNA (scramble, CDH2, or Cx43). Bottom panel: normalized band densities. (H, I, J) Dye transfer assay: M-Oct4-GFP/scramble-RFP, M-Oct4-GFP/CDH2-shRNA-RFP, and M-Oct4 GFP/Cx43-shRNA-RFP were co-cultured at 1:1 ratio with CMAC-blue–labeled BM-stroma (H) or BM-MSCs (I) in four well chamber slides. After 72 h, the cells were imaged for dye transfer. Negative control used co-cultures of M-Oct4-GFP/scramble-RFP and CMAC-blue–labeled stroma or MSCs in the presence of 300 µM 1-octanol. The white spots (white arrows) in the vehicle-treated M-Oct4-GFP/scramble-RFP indicate dye transfer. Inset: enlarged image depicts cells with dye transfer. The cells were imaged using EVOS FL2 Auto 2 at 200× magnification. (K, L) CDH2-shRNA and Cx43 shRNA MDA-MB-231 were infected with inducible lentiviral particle pLVX-CDH2/GS and pLVX-Cx43/GS, respectively. After overnight infection, the cells were induced with different concentrations of doxocycline: 0, 5, 10, 100, 500, and 1,000 ng/ml. After 24 h, proteins were isolated and Western blot performed for CDH2 and Cx43. The membranes were stripped and reprobed with anti–β-actin. Source data are available for this figure.

Journal: Life Science Alliance

Article Title: Specific N-cadherin–dependent pathways drive human breast cancer dormancy in bone marrow

doi: 10.26508/lsa.202000969

Figure Lengend Snippet: (A) Representative image of Passage 3 mesenchymal stem cells (MSCs), 400× magnification. (B) Flow cytometry represents BM-MSC characterization. Cells were labeled with anti–CD73-FITC, anti–CD29-PE, and anti–CD90-PerCP-Cy5.5. (C) Induced differentiation of BM-MSCs was performed by culturing the cells in maintenance media or differentiation media (osteogenic or adipogenic). After week 3, cells in the osteocytic media were counter stained with Von Kossa to assess calcium deposit. Representative images are shown for osteocytes (top) and adipocyte (bottom). (D) Whole-cell lysates from BM-MSCs or BM stromal cells were immunoprecipitated with anti-CDH2 or IgG (Antibody control) and then electrophorese on 12% SDS–PAGE. The proteins were transferred onto PVDF membrane for blotting with anti-Cx43. (E) Real time PCR for CDH2. The results were normalized for β-actin and then presented as the mean ± SD (n = 5) relative expression. (F) Western blot with extracts from BC cell subsets: Oct4a-GFP hi, med or lo . (G, H) Top panel: Western blot for CDH2 (F) and Cx43 (G) using whole-cell extracts from MDA-MB-231, transfected with shRNA (scramble, CDH2, or Cx43). Bottom panel: normalized band densities. (H, I, J) Dye transfer assay: M-Oct4-GFP/scramble-RFP, M-Oct4-GFP/CDH2-shRNA-RFP, and M-Oct4 GFP/Cx43-shRNA-RFP were co-cultured at 1:1 ratio with CMAC-blue–labeled BM-stroma (H) or BM-MSCs (I) in four well chamber slides. After 72 h, the cells were imaged for dye transfer. Negative control used co-cultures of M-Oct4-GFP/scramble-RFP and CMAC-blue–labeled stroma or MSCs in the presence of 300 µM 1-octanol. The white spots (white arrows) in the vehicle-treated M-Oct4-GFP/scramble-RFP indicate dye transfer. Inset: enlarged image depicts cells with dye transfer. The cells were imaged using EVOS FL2 Auto 2 at 200× magnification. (K, L) CDH2-shRNA and Cx43 shRNA MDA-MB-231 were infected with inducible lentiviral particle pLVX-CDH2/GS and pLVX-Cx43/GS, respectively. After overnight infection, the cells were induced with different concentrations of doxocycline: 0, 5, 10, 100, 500, and 1,000 ng/ml. After 24 h, proteins were isolated and Western blot performed for CDH2 and Cx43. The membranes were stripped and reprobed with anti–β-actin. Source data are available for this figure.

Article Snippet: Mouse anti-human CDK4 mAb (1/1,000 dilution), anti-human CDK6 mAb (1/1,000 dilution), mouse anti-human cyclin E mAb (1/1,000 dilution), rabbit anti-human cyclin D1 mAb (1/1,000 dilution), rabbit anti-human Cx43 (1/1,000 dilution), rabbit anti-human Vimentin (1/1,000 dilution), rabbit anti-human Slug (1/1,000 dilution), rabbit anti-human β-catenin (1/1,000 dilution), rabbit anti-human Zeb1 (1/1,000 dilution), rabbit anti-human caspase 3 (1/1,000 dilution), rabbit anti-human caspase 7 (1/1,000 dilution), rabbit anti-human MDR1 (1/1,000 dilution), rabbit anti-human E-cadherin (1/1,000 dilution), rabbit anti-human p38 (1/1,000 dilution), rabbit anti-human p-p38 (1/1,000 dilution), rabbit anti-human Erk1/2 (1/1,000 dilution), rabbit anti-human p-Erk1/2 (1/1,000 dilution), and normal anti-rabbit immunoglobulin G (IgG) were purchased from Cell Signaling Technology; rabbit polyclonal anti-human cyclin A (1/1,000 dilution), and goat anti-rabbit IgG-TR were purchased from Santa Cruz Biotechnology; rabbit polyclonal anti-human CDH2 (1/1,000 dilution), rabbit polyclonal anti-human Ki67 (1/200 dilution), and rabbit anti-human vinculin mAb (1/1,000 dilution) from Abcam; rabbit anti-human Twist (1/1,000 dilution), mouse anti-human pan-cytokeratin (1/200 dilution), and mouse anti-human β-actin (1/2,000 dilution) from Millipore Sigma; goat polyclonal anti-rabbit IgG-HRP (1/5,000 dilution), goat polyclonal anti-murine IgG-HRP (1/5,000 dilution), goat anti-rabbit IgG–Alexa Fluor 405 (1/500 dilution), goat anti-mouse IgG–Alexa Fluor 488 (1/500 dilution), goat anti-rabbit IgG-Alexa Fluor 610 (1/500 dilution), goat anti-mouse Alexa 647 IgG (1/500 dilution), rabbit anti-human HA tag (1/1,000 dilution), rabbit anti-human Flag tag (1/1,000 dilution), goat polyclonal anti-rabbit IgG-AP (1/500 dilution), rabbit anti-human GM130 (1/1,000 dilution), rabbit anti-human GRP78 (1/1,000 dilution), and CD14 + Dynabeads Flow kit from Thermo Fisher Scientific; HRP-mouse anti-rabbit IgG light chain–specific from Jackson Immuno Research, murine anti-CD206-PE (1/20 dilution), murine anti-HLA-DR-APC (1/20 dilution), murine anti-human CDH2-PE (1/20 dilution), FITC murine anti-human CD73 (1/20 dilution), PE murine anti-human CD29 (1/20 dilution), murine anti-human CD90 − perCp/Cyanine 5.5 (1/20 dilution) from BD Biosciences; and recombinant human M-CSF, and IL-4 from R & D Systems.

Techniques: Flow Cytometry, Labeling, Staining, Immunoprecipitation, SDS Page, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, shRNA, Cell Culture, Negative Control, Infection, Isolation

Effects of the CD73 inhibitor, α,β‐methylene ADP (AOPCP, 100 μM administered intracerebroventricularly at 0.25 μl·hr −1 for 14 days) on the decreased levels of dopamine and its metabolite (ng·mg −1 tissue) in the rat striatum and mesencephalon after 6‐OHDA administration

Journal: British Journal of Pharmacology

Article Title: Enhanced ATP release and CD73‐mediated adenosine formation sustain adenosine A 2A receptor over‐activation in a rat model of Parkinson's disease

doi: 10.1111/bph.14771

Figure Lengend Snippet: Effects of the CD73 inhibitor, α,β‐methylene ADP (AOPCP, 100 μM administered intracerebroventricularly at 0.25 μl·hr −1 for 14 days) on the decreased levels of dopamine and its metabolite (ng·mg −1 tissue) in the rat striatum and mesencephalon after 6‐OHDA administration

Article Snippet: The analysis of CD73 and A 2A receptor density was carried out as previously described (Augusto et al., 2013 ) using mouse anti‐A 2A R (1:1,000, Merck‐Millipore, clone 7F6‐G5‐A2), and goat anti‐murine CD73 (1:1,000, Santa Cruz Biotechnology, sc‐25603), followed by the appropriate secondary antibody conjugated with alkaline phosphatase (Amersham) before revealing the membranes with ECF (Amersham).

Techniques:

The unilateral administration of 6‐OHDA in the striatum of rats increases the evoked release of ATP from depolarized synapses, up‐regulates ecto‐5′‐nucleotidase (CD73) selectively in synapses, enhancing the formation of extracellular adenosine (ADO) from adenine nucleotides. (a) The high K+‐evoked release of ATP from striatal synaptosomes was greater in synaptosomes derived from mice killed 19 days after being challenged with 6‐OHDA intra‐striatally, compared with saline treated (control). (b) The density of immuno‐reactive CD73 was selectively enhanced in membranes from synaptosomes (synap), but not in total membranes (TMs), from 6‐OHDA (6OH)‐treated rats, compared with those from saline (sal)‐treated rats. (c) The extracellular catabolism of AMP (10 μM, added at Time 0) and the formation of ADO were greater in striatal synaptosomes from 6‐OHDA‐treated rats than in those from saline‐treated rats. (d) α,β‐Methylene ADP (AOPCP, 100 μM) blocked the extracellular catabolism of AMP (10 μM, added at Time 0) into adenosine in synaptosomes from 6‐OHDA‐treated rats. The data shown are means ± SEM of n = 4–5. *P < .05, significantly different from control; Dunnett's test in (a) and significantly different from saline; two‐tailed unpaired Student's t test

Journal: British Journal of Pharmacology

Article Title: Enhanced ATP release and CD73‐mediated adenosine formation sustain adenosine A 2A receptor over‐activation in a rat model of Parkinson's disease

doi: 10.1111/bph.14771

Figure Lengend Snippet: The unilateral administration of 6‐OHDA in the striatum of rats increases the evoked release of ATP from depolarized synapses, up‐regulates ecto‐5′‐nucleotidase (CD73) selectively in synapses, enhancing the formation of extracellular adenosine (ADO) from adenine nucleotides. (a) The high K+‐evoked release of ATP from striatal synaptosomes was greater in synaptosomes derived from mice killed 19 days after being challenged with 6‐OHDA intra‐striatally, compared with saline treated (control). (b) The density of immuno‐reactive CD73 was selectively enhanced in membranes from synaptosomes (synap), but not in total membranes (TMs), from 6‐OHDA (6OH)‐treated rats, compared with those from saline (sal)‐treated rats. (c) The extracellular catabolism of AMP (10 μM, added at Time 0) and the formation of ADO were greater in striatal synaptosomes from 6‐OHDA‐treated rats than in those from saline‐treated rats. (d) α,β‐Methylene ADP (AOPCP, 100 μM) blocked the extracellular catabolism of AMP (10 μM, added at Time 0) into adenosine in synaptosomes from 6‐OHDA‐treated rats. The data shown are means ± SEM of n = 4–5. *P < .05, significantly different from control; Dunnett's test in (a) and significantly different from saline; two‐tailed unpaired Student's t test

Techniques: Derivative Assay, Two Tailed Test

The elimination of ATP‐derived formation of extracellular adenosine abrogates 6‐OHDA‐induced dopaminergic neurodegeneration in the striatum and nigra. Representative coronal sections of the dorsolateral striatum (a) or substantia nigra (c) stained TH from saline‐treated rats and 6‐OHDA‐treated rats without or with the CD73 inhibitor α,β‐methylene ADP (AOPCP, 100 μM continuous intracerebroventricular infusion). Immunohistochemical quantification of TH density in the striatum (b) or substantia nigra (d). The data are means ± SEM (n = 6). *P < .05, significantly different from saline; # P < .05, significantly different from 6‐OHDA; one‐way ANOVA with Newman–Keuls post hoc test

Journal: British Journal of Pharmacology

Article Title: Enhanced ATP release and CD73‐mediated adenosine formation sustain adenosine A 2A receptor over‐activation in a rat model of Parkinson's disease

doi: 10.1111/bph.14771

Figure Lengend Snippet: The elimination of ATP‐derived formation of extracellular adenosine abrogates 6‐OHDA‐induced dopaminergic neurodegeneration in the striatum and nigra. Representative coronal sections of the dorsolateral striatum (a) or substantia nigra (c) stained TH from saline‐treated rats and 6‐OHDA‐treated rats without or with the CD73 inhibitor α,β‐methylene ADP (AOPCP, 100 μM continuous intracerebroventricular infusion). Immunohistochemical quantification of TH density in the striatum (b) or substantia nigra (d). The data are means ± SEM (n = 6). *P < .05, significantly different from saline; # P < .05, significantly different from 6‐OHDA; one‐way ANOVA with Newman–Keuls post hoc test

Techniques: Derivative Assay, Staining, Immunohistochemical staining

Blocking the formation of extracellular adenosine derived from ATP prevents 6‐OHDA‐induced motor and memory dysfunction. (a) The continuous intracerebroventricular infusion of the CD73 inhibitor α,β‐methylene ADP (AOPCP, 100 μM) attenuated the apomorphine‐induced contralateral rotation in rats unilaterally challenged with 6‐OHDA. (b) The unilateral injection of 6‐OHDA decreased the use of the contralateral limb, especially together with the ipsilateral limb, and this was restored after treatment with AOPCP (100 μM continuous intracerebroventricular infusion). (c) Neither the unilateral injection of 6‐OHDA nor the treatment with AOPCP altered the spontaneous locomotion in the open field. (d) AOPCP also prevented the decreased recognition memory of rats intra‐striatally injected with 6‐OHDA, as evaluated in the novel object recognition task: All groups of rats investigated equally both objects in the training phase (not shown), but only 6‐OHDA‐lesioned rats failed to discriminate the novel object during the test phase (e), without alteration of the total distance travelled during the test session between the four groups (not shown). Data shown are means ± SEM (n = 10 rats per group). *P < .05, significantly different from saline; **P < .05 significantly different from 6‐OHDA; # P < .05 significantly different from saline; two‐way ANOVA with Newman–Keuls post hoc test

Journal: British Journal of Pharmacology

Article Title: Enhanced ATP release and CD73‐mediated adenosine formation sustain adenosine A 2A receptor over‐activation in a rat model of Parkinson's disease

doi: 10.1111/bph.14771

Figure Lengend Snippet: Blocking the formation of extracellular adenosine derived from ATP prevents 6‐OHDA‐induced motor and memory dysfunction. (a) The continuous intracerebroventricular infusion of the CD73 inhibitor α,β‐methylene ADP (AOPCP, 100 μM) attenuated the apomorphine‐induced contralateral rotation in rats unilaterally challenged with 6‐OHDA. (b) The unilateral injection of 6‐OHDA decreased the use of the contralateral limb, especially together with the ipsilateral limb, and this was restored after treatment with AOPCP (100 μM continuous intracerebroventricular infusion). (c) Neither the unilateral injection of 6‐OHDA nor the treatment with AOPCP altered the spontaneous locomotion in the open field. (d) AOPCP also prevented the decreased recognition memory of rats intra‐striatally injected with 6‐OHDA, as evaluated in the novel object recognition task: All groups of rats investigated equally both objects in the training phase (not shown), but only 6‐OHDA‐lesioned rats failed to discriminate the novel object during the test phase (e), without alteration of the total distance travelled during the test session between the four groups (not shown). Data shown are means ± SEM (n = 10 rats per group). *P < .05, significantly different from saline; **P < .05 significantly different from 6‐OHDA; # P < .05 significantly different from saline; two‐way ANOVA with Newman–Keuls post hoc test

Techniques: Blocking Assay, Derivative Assay, Injection

6‐Hydroxydopamine (6‐OHDA, 30 μM) leads to a rapid and sustained increase of extracellular ATP and an up‐regulation of both A2A receptors and of the ecto‐5′‐nucleotidase (CD73) responsible for an increased formation of extracellular adenosine (ADO) derived from adenine nucleotides in differentiated SY5Y neuroblastoma cells. (a) The extracellular levels of ATP were rapidly (within 5 min) increased in a sustained manner (up to 6 hr) after the exposure to 6‐OHDA. (b, c) Western blot analysis showed an increased density of immuno‐reactive A2A receptors (A2AR) and of CD73 but not of the ecto‐nucleoside triphosphate diphosphohydrolase‐1 (NTPDase1 / CD39) at 2 hr after adding 6‐OHDA. (d) The extracellular catabolism of AMP (10 μM, added at Time 0) and the formation of adenosine were greater in cells exposed for 2 hr to 6‐OHDA, compared those when cells were exposed to Krebs solution only. (e) α,β‐Methylene ADP (AOPCP, 100 μM) blocked the extracellular hydrolysis of AMP (10 μM, added at Time 0) to adenosine. (f, g) The extracellular catabolism of ATP (10 μM, added at Time 0) was similar in the control (f) and 6‐OHDA‐treated cells (g), but the formation of extracellular AMP was lower and the formation of ADO was higher in cells exposed for 2 hr to 6‐OHDA (g) compared with data from cells exposed to Krebs solution only (f). Except for the representative Western blots (b), the data shown are means ± SEM of n = 5–10 in (a), n = 5–6 in (c), n = 4 (d), n = 3 (e), and n = 4 (f, g). *P < .05, significantly different from control; Dunnett's test in (a) and a two‐tailed unpaired Student's t test in (c)

Journal: British Journal of Pharmacology

Article Title: Enhanced ATP release and CD73‐mediated adenosine formation sustain adenosine A 2A receptor over‐activation in a rat model of Parkinson's disease

doi: 10.1111/bph.14771

Figure Lengend Snippet: 6‐Hydroxydopamine (6‐OHDA, 30 μM) leads to a rapid and sustained increase of extracellular ATP and an up‐regulation of both A2A receptors and of the ecto‐5′‐nucleotidase (CD73) responsible for an increased formation of extracellular adenosine (ADO) derived from adenine nucleotides in differentiated SY5Y neuroblastoma cells. (a) The extracellular levels of ATP were rapidly (within 5 min) increased in a sustained manner (up to 6 hr) after the exposure to 6‐OHDA. (b, c) Western blot analysis showed an increased density of immuno‐reactive A2A receptors (A2AR) and of CD73 but not of the ecto‐nucleoside triphosphate diphosphohydrolase‐1 (NTPDase1 / CD39) at 2 hr after adding 6‐OHDA. (d) The extracellular catabolism of AMP (10 μM, added at Time 0) and the formation of adenosine were greater in cells exposed for 2 hr to 6‐OHDA, compared those when cells were exposed to Krebs solution only. (e) α,β‐Methylene ADP (AOPCP, 100 μM) blocked the extracellular hydrolysis of AMP (10 μM, added at Time 0) to adenosine. (f, g) The extracellular catabolism of ATP (10 μM, added at Time 0) was similar in the control (f) and 6‐OHDA‐treated cells (g), but the formation of extracellular AMP was lower and the formation of ADO was higher in cells exposed for 2 hr to 6‐OHDA (g) compared with data from cells exposed to Krebs solution only (f). Except for the representative Western blots (b), the data shown are means ± SEM of n = 5–10 in (a), n = 5–6 in (c), n = 4 (d), n = 3 (e), and n = 4 (f, g). *P < .05, significantly different from control; Dunnett's test in (a) and a two‐tailed unpaired Student's t test in (c)

Techniques: Derivative Assay, Western Blot, Two Tailed Test

Bar graphs show levels of HSCs (Sca1 + cKit + CD31 − ), MSCs (Sca1 + CD105 + CD31 − ), and EPCs (Sca1 + ckit + CD31 + ) in peripheral blood of db/db and db/m mice. * p < 0.05 compared to the db/m group

Journal: The EPMA Journal

Article Title: Status of stem cells in diabetic nephropathy: predictive and preventive potentials

doi: 10.1186/s13167-016-0070-6

Figure Lengend Snippet: Bar graphs show levels of HSCs (Sca1 + cKit + CD31 − ), MSCs (Sca1 + CD105 + CD31 − ), and EPCs (Sca1 + ckit + CD31 + ) in peripheral blood of db/db and db/m mice. * p < 0.05 compared to the db/m group

Article Snippet: Antibodies against Sca1, cKit, CD105, CD73, CD31 (markers of murine stem cells), and caspase 3 (for apoptosis) were obtained from BD Biosciences (San Diego, CA).

Techniques:

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murine cd73 (AMS Biotechnology)

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