Journal: Bioactive Materials

Article Title: Immunomodulatory supramolecular hydrogel for rheumatoid arthritis management via adenosine A2A receptor-mediated macrophage remodeling

doi: 10.1016/j.bioactmat.2025.11.031

Figure Lengend Snippet: The design of an inflammatory macrophages-targeted, acid-sensitive PNSH for delivery MTX and its role in promoting A2AR activation on M2 macrophages for arthritis therapy. (A)The structure of acid-sensitive polymer-nanomedicine supramolecular hydrogels, composed of drug-loaded MTX NPs. (B) Schematic illustration of arthritis therapy targeting inflammatory joint network via mPECN NPs-mediated release of MTX. This approach leverages MTX targeting the A2AR and repolarization of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Activation of A2AR and macrophage repolarization by mPECN-MTX NPs synergistically enhanced the anti-inflammatory effect of PNSH-mediated nanomedicine in an arthritis rat model.

Article Snippet: Subsequently, 10 mg of protein was loaded into 10 % SDS-polyacrylamide gel electrophoresis for detection of adenosine A2AR (sc-32261, Santa Cruz), IRF4 (#62834S, Cell Signaling Technology, MA, USA), IRF8 (#5628T, Cell Signaling Technology, MA, USA) and GAPDH (TA-08, ZSGB-BIO, Beijing, China).

Techniques: Activation Assay, Polymer

Journal: Bioactive Materials

Article Title: Immunomodulatory supramolecular hydrogel for rheumatoid arthritis management via adenosine A2A receptor-mediated macrophage remodeling

doi: 10.1016/j.bioactmat.2025.11.031

Figure Lengend Snippet: MTX@PNSH promotes anti-inflammatory M2 macrophages through the A2AR signaling pathway. (A) The intracellular cAMP levels in Raw 264.7 cells were determined by homogeneous time-resolved fluorescence (HTRF) following different treatments. (B) qRT-PCR analysis of relative mRNA of A2ar with different treatments. (C) Immunostaining of A2AR expression in Raw 264.7 cells with different treatments. (D) Flow cytometry analysis of A2AR + cells in Raw 264.7 cells with different treatments and the percentage of A2AR + cells in Raw 264.7 cells with different treatments. (E) qRT-PCR analysis of relative mRNA expression of Pdl1 without LPS treatment. (F) qRT-PCR analysis of relative mRNA expression of Ido1 without LPS treatment. (G) qRT-PCR analysis of relative mRNA expression of Pdl1 with LPS treatment. (H) qRT-PCR analysis of relative mRNA expression of Ido1 with LPS treatments. (I) Western blotting analysis of A2AR protein level in macrophages after 24 h of different treatment with or without A2AR inhibitor. (J) Quantitative analysis of A2AR protein level in different treatments with or without A2AR inhibitor. (K) Flow cytometry analysis of CD11b and CD39 in Raw 264.7 cells with different treatments and the percentage of CD11b + CD39 + macrophages. (L) Flow cytometry analysis of CD11b and CD73 in Raw 264.7 cells with different treatments and the percentage of CD11b + CD73 + macrophages. (M) Flow cytometry analysis of CD11b and PD-L1 in Raw 264.7 cells after 24 h of different treatments and the percentage of CD11b + PD-L1 + macrophages in Raw 264.7 cells after 24 h of different treatments. Data are presented as mean ± SD, n = 3. Statistical significance was determined using one-way ANOVA, followed by Dunnett's post hoc test for comparisons between groups.

Article Snippet: Subsequently, 10 mg of protein was loaded into 10 % SDS-polyacrylamide gel electrophoresis for detection of adenosine A2AR (sc-32261, Santa Cruz), IRF4 (#62834S, Cell Signaling Technology, MA, USA), IRF8 (#5628T, Cell Signaling Technology, MA, USA) and GAPDH (TA-08, ZSGB-BIO, Beijing, China).

Techniques: Fluorescence, Quantitative RT-PCR, Immunostaining, Expressing, Flow Cytometry, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: PBF-509 is specific for the A2aR. (A) Dose response curves of PBF-509 versus 4 adenosine receptors in transfected CHO (hA1), HeLa (hA2a and hA3), and HEK-293 (hA2b) cells. (B) Representative dose–response curves of PBF-509 in a binding assay against the human A2aR in buffer (median Ki = 12 nM) and in human plasma (median Ki = 32 nM).

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Transfection, Binding Assay, Clinical Proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: PBF-509 inhibits accumulation of cAMP. CHO cells transfected with the A2aR were used to determine the concentration of PBF-509 to reduce the concentration of cAMP by 50%.

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Transfection, Concentration Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: Human primary tumor, CAF, and TIL cell lines express CD73 and A2aR. Tumor (Tm), cancer-associated fibroblast (CAF), and tumor infiltrating lymphocyte (TIL) cell lines were established from 12 resected NSCLC tumors. (A) Tumor cells or CAFs were stained with anti-CD73 and subjected to flow cytometry. Most cells from nearly all of the cell lines (both tumor cells and CAFs) expressed high levels of CD73. (B) Representative experiment of tumor cells stained with anti-CD73 (blue) or isotype control (green). (C) CD4+ or CD8+ TIL cells were stained with anti-A2aR and subjected to flow cytometric analysis showing that high levels of A2aR were being expressed in CD4+ cells, with variable expression in CD8+ cells.

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Staining, Flow Cytometry, Control, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: A Novel Antagonist of the Immune Checkpoint Protein Adenosine A2a Receptor Restores Tumor-Infiltrating Lymphocyte Activity in the Context of the Tumor Microenvironment

doi: 10.1016/j.neo.2017.02.004

Figure Lengend Snippet: Anti-PD-L1 and PBF-509 restore immune responsiveness of TILs. Four different resected NSCLC tumors were disaggregated and cultured with or without anti-PD-L1 (aPDL1; 10 mg/ml), anti-PD-1 (aPD1; 10 μg/ml), or the A2aR antagonist PBF-509 (1 μM). After 3 days in culture, the supernatants were assayed for the presence of γ-interferon using an ELISA.

Article Snippet: After tumor disaggregation, the cells were stained for Live/Dead NearIR (Life Technologies; L10119), CD3 (Fisher Scientific; BDB563546), CD4 (Fisher Scientific; BDB562970), CD8 (Fisher Scientific; BDB562282), and A2aR (Novus Biologicals; NBP1-39474FR) to determine the percentage of A2aR-positive cells in CD4+ and CD8+ cells.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Baseline expression level of inhibitory receptors in T cells of patients with EOC and healthy controls. The baseline percentage of TIM3+ (A) , PD1+ (B) , and A2aR+ (C) cells are significantly higher in EOC patient-derived T cells post thawing. The frequency of T tex cells is significantly higher in patients’ samples compared to normal controls (D) . However, the frequency of T pex cells is not significant different between patients and healthy samples (D). Representative overlay histogram plots representing expression level of TIM3 (A), PD1 (B), and A2aR (C) in EOC patient-derived T cells sample (Red color) vs. unstained sample (Blue color). Representative overlay dot plot (D) representing the frequency of terminally exhausted and progenitor exhausted T cells in an EOC patient sample (Red color) vs. unstained sample (Blue color). Data from healthy donors (n = 30) and EOC patients (n = 30) were analyzed using Welch’s t-test (A and B) Mann-Whitney U test (C and D). * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Derivative Assay, MANN-WHITNEY

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Expression pattern of inhibitory receptors during CAR T manufacturing process. CAR T cell population positive for TIM3 (A) , PD1 (B) , and A2aR (C) in EOC patient-derived T cells during CAR T cell manufacturing steps. The frequency of T pex (D) and T tex (E) cells during manufacturing steps of CAR T cells derived from EOC patient T cells. Median fluorescence intensity (MFI) of A2aR (F) , TIM3 (G) , and PD1 (H) in EOC patient-derived T cells during CAR T cells manufacturing process. Healthy donors (n = 30) and EOC patients (n = 30) data were analyzed using RM one-way ANOVA and Tukey multiple comparison test. * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Derivative Assay, Fluorescence, Comparison

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Expression pattern of inhibitory receptors in patient-derived CAR T cells. CAR T cell population positive for TIM3 (A) , PD1 (B) , and A2aR (C) in EOC patient-derived T cells after activation and transduction vs. healthy controls. The frequency of T tex (D) and T pex (E) cells after activation and transduction of EOC patient-derived T cells vs. healthy controls. Data from healthy donors (n = 30) and EOC patients (n = 30) were analyzed using Mann-Whitney U test (A, B, and D) and Welch’s t-test (C and E). * P < 0.05; **P < 0.01; ***P < 0.001. Data are represented by mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Derivative Assay, Activation Assay, Transduction, MANN-WHITNEY

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Differential expression of immune inhibitory receptors in different cancer stages during CAR T cell manufacturing. Cell surface expression levels of TIM3 (A) , PD1 (B) , A2aR (C) , and the frequency of T pex (D) and T tex (E) cells at different steps of CAR T cell manufacturing process for different stages of EOC. Data from stage III patients (n = 18) and stage IV patients (n = 12) were analyzed using Welch’s t-test. * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented by mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Quantitative Proteomics, Expressing

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Effect of CarboTaxol treatment on T cell expansion, frequency, and expression pattern of immune inhibitory receptors during CAR T cell manufacturing process. Fold increase of T cells from CarboTaxol-treated and untreated patients post T cell activation (A) . CD3 expression after T cell activation in CarboTaxol treated and newly diagnosed patients (B) . The percentage of A2aR (C) , TIM3 (D) , PD1 (E) positive cells, and the frequency of T tex (F) and T pex cells (G) during CAR T cell manufacturing process in CarboTaxol-treated and newly diagnosed patients. Data from CarboTaxol-treated patients (n = 20) and new patients (n = 10) were analyzed using Welch’s t-test. * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented by mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Activation Assay

Journal: Cell biochemistry and function

Article Title: Inhibition of CD73 expression or A2AR blockade reduces MRP1 expression and increases the sensitivity of cervical cancer cells to cisplatin.

doi: 10.1002/cbf.3784

Figure Lengend Snippet: FIGURE 1 Effect of ADO and blockade of ARs on MRP1 expression in CC cells. (A) CaSki and HeLa cells, 1 × 105 of each, were cultured for 48 h with different concentrations of ADO (0, 0.001, 0.01, 0.1, and 1 mM), and MRP1 expression was analyzed by flow cytometry. The mean fluorescence intensity of MRP1 in cells cultured in the absence of ADO was normalized to 1. AF, Autofluorescence. Representative histograms from three independent experiments. (B) MRP1 expression in CaSki and HeLa cells cultured in the presence of 1 mM ADO and presence (+) or absence (−) of 10 μM DPCPX, ZM241385, MRS1754, and MRS1523, specific antagonists of A1R, A2AR, A2BR, and A3R, respectively, which are indicated in parentheses. Representative data from three independent experiments ± SEM. *p < .05, one‐way ANOVA.

Article Snippet: The expression of CD73, MRP1, A1R, A2AR, A2BR, and A3R in CaSki and HeLa cell lines was determined by flow cytometry using the following antibodies: CD73‐PE (PHA550257, BD Pharmingen), anti‐ MRP1 (MRPM5, Abcam), A1R‐AF647 (s‐4235R‐A647, Bioss), A2AR‐ PE (NBP1‐39474PE, Novus Biologicals), A2BR‐AF750 (s‐5900R‐ A750, Bioss), and A3R‐FITC (orb399642, Biorbyt).

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence

Journal: Cell biochemistry and function

Article Title: Inhibition of CD73 expression or A2AR blockade reduces MRP1 expression and increases the sensitivity of cervical cancer cells to cisplatin.

doi: 10.1002/cbf.3784

Figure Lengend Snippet: FIGURE 3 CD73 inhibition or A2AR blockade in CC cells decreases the extrusive capacity of CC cells. (A) CaSki‐WT and CaSki‐pS‐siRNA‐ CD73 cells; (B) CaSki‐WT and CaSki‐ADO + ZM241385 cells; (C) HeLa‐WT and HeLa‐pS‐siRNA‐CD73 cells; and (D) HeLa‐WT and HeLa‐ ADO + ZM241385 cells, 5 × 105 each, were cultured in the presence of different concentrations (0, 10, 20, 40, 60, 80, and 100 μM) of MK‐751, a specific inhibitor of MRP1, and the fluorogenic dye calcein‐AM, a substrate of MRP1, as described in the materials and methods section. The intracellular calcein‐AM content in CaSki‐WT and HeLa‐WT cells in the absence of MK‐751 was normalized to 100%. Representative data from three independent experiments ± SEM. *p < .05; **p < .01; ***p < .001, and ****p < .0001, one‐way ANOVA.

Article Snippet: The expression of CD73, MRP1, A1R, A2AR, A2BR, and A3R in CaSki and HeLa cell lines was determined by flow cytometry using the following antibodies: CD73‐PE (PHA550257, BD Pharmingen), anti‐ MRP1 (MRPM5, Abcam), A1R‐AF647 (s‐4235R‐A647, Bioss), A2AR‐ PE (NBP1‐39474PE, Novus Biologicals), A2BR‐AF750 (s‐5900R‐ A750, Bioss), and A3R‐FITC (orb399642, Biorbyt).

Techniques: Inhibition, Cell Culture

Journal: Cell biochemistry and function

Article Title: Inhibition of CD73 expression or A2AR blockade reduces MRP1 expression and increases the sensitivity of cervical cancer cells to cisplatin.

doi: 10.1002/cbf.3784

Figure Lengend Snippet: FIGURE 4 Effect of CP on the viability of CC cells with downregulated CD73 expression or in which A2AR was blocked. The effect of different concentrations of CP (0, 3.12, 6.25, 12.5, 25, 50, and 100 μM) was evaluated on the viability of (A) parental CaSki and HeLa cells (WT), cells transfected with pSIREN vector (pSIREN), or cells transfected with pSIREN vector containing a siRNA targeting CD73 (pS/siRNA‐CD73); (B) parental CaSki and HeLa cells (WT), treated with 1 mM ADO (ADO) or treated with ADO in the presence of 10 µM A2AR antagonist (ADO + ZM241385); and c) parental CaSki and HeLa cells (WT) or CaSki and HeLa cells treated with 100 µM MK‐751 for 24 h, as described in the materials and methods section. Representative data from three independent experiments. CP IC50 values for each cell type were calculated using GraphPad Prism version 8 using log‐transformed and normalized OD values and CP concentrations (μM) from three experiments. *p < .05; ***p < .001, and ****p < .0001, one‐way ANOVA.

Article Snippet: The expression of CD73, MRP1, A1R, A2AR, A2BR, and A3R in CaSki and HeLa cell lines was determined by flow cytometry using the following antibodies: CD73‐PE (PHA550257, BD Pharmingen), anti‐ MRP1 (MRPM5, Abcam), A1R‐AF647 (s‐4235R‐A647, Bioss), A2AR‐ PE (NBP1‐39474PE, Novus Biologicals), A2BR‐AF750 (s‐5900R‐ A750, Bioss), and A3R‐FITC (orb399642, Biorbyt).

Techniques: Expressing, Transfection, Plasmid Preparation, Transformation Assay

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Synergistic blockade of SHP-2 and A2AR signal pathways with targeted nanoparticles restores anti-tumor immunity of CD8+ T cells.

doi: 10.1016/j.jconrel.2025.113889

Figure Lengend Snippet: Scheme 1. Schematic illustration of T cell anti-tumor responses promoted by targeting nanoparticles loaded with SHP2 and A2AR inhibitors. (a) Schematic preparation of T cell-targeting nanoparticles. (b) Schematic presentation of activation of SHP2/PD-1 and adenosine/A2AR pathways resulting in T cell exhaustion and cancer immune escapes. (c) Schematic showcase of delivery of SHP099 and CPI-444 to the T cell leading to concurrent blockade of A2AR and SHP2 signaling pathways, rescuing T cell functions.

Article Snippet: Alexa Fluor® 647-conjugated anti-human A2AR antibody was bought from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Protein-Protein interactions

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Synergistic blockade of SHP-2 and A2AR signal pathways with targeted nanoparticles restores anti-tumor immunity of CD8+ T cells.

doi: 10.1016/j.jconrel.2025.113889

Figure Lengend Snippet: Fig. 4. SCNP/αCD8 restored T-cell activity in the presence of immune suppression. (a) PD-1 expression on human CD8+ T cells following activation with αCD3/CD28 beads complex. (b) A2AR expression on CD8+ T cells following activation with anti-CD3/CD28 complex. Flow cytometry assay of T cells positive with CD107a (c) and CD69 (d) when αCD3/CD28 activated CD8+ T cell co-cultured with CGS-21680 and peptide (10 mM) in the present or absence dual-drug loaded untargeted, isotype and targeted nanoparticles. Data are shown as mean ± SD, n = 3. ****p < 0.0001.

Article Snippet: Alexa Fluor® 647-conjugated anti-human A2AR antibody was bought from R&D systems (Minneapolis, MN).

Techniques: Activity Assay, Expressing, Activation Assay, Flow Cytometry, Cell Culture

Journal: Circulation Research

Article Title: Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells

doi: 10.1161/01.res.0000165800.81876.52

Figure Lengend Snippet: Figure 2. AR mRNA levels in cultured CASMCs and intact coro- nary arteries. RT-PCR for detection of the A1R, A2AR (A) and A3R (B) mRNA expression was performed with cultured CASMCs or CASMCs dispersed from freshly isolated porcine right coronary arteries (RCA) denuded of endothelium. Primary cultured por- cine coronary artery endothelial cells (CAEC, B) were used as controls. RT-PCR performed without reverse transcriptase is indicated as “RT.”

Article Snippet: Cells were lysed and lysates Western blotted for the A1R (rabbit polyclonal antibody, 1:1000 dilutions, Novus Biologicals, Inc).

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Reverse Transcription

Journal: Circulation Research

Article Title: Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells

doi: 10.1161/01.res.0000165800.81876.52

Figure Lengend Snippet: Figure 5. Sequence, pharmacology, and signaling of the porcine A1R. Deduced amino acid sequence of the cloned A1R was aligned with the human homolog (A). Putative transmembrane-spanning domains are indicated with solid lines and marked as TM I to TM VII. Sequence matches are shaded. F, Potential phosphorylation sites; Œ, consensus sites for N-linked glycosylation; and *Putative palmi- toylation site (A). Saturation binding of [3H]-DPCPX was performed on membranes from stably transfected COS-1 cells or cultured CASMCs (B). Only specific binding is shown. Each point is the mean of triplicate determinations. Binding affinities of various ligands were determined from the competition binding assays on the membranes of transfected COS-1 cells in the presence of 0.5 nmol/L [3H]-DPCPX (C). Data shown are the meanSEM from 3 experiments performed in triplicate. Inhibition of forskolin-stimulated cAMP production by CCPA in transfected COS-1 cells (f) and in cultured CASMCs (▫) (D). Increase in [Ca2]i by CCPA was detected only in transfected COS-1 cells (E). Data for cAMP assay are the meanSEM from 3 independent experiments performed in duplicate and data points in the Ca2 curve indicate the average peak increase in [Ca2]i for 8 to 16 cells on each of 5 coverslips and represent the meanSEM.

Article Snippet: Cells were lysed and lysates Western blotted for the A1R (rabbit polyclonal antibody, 1:1000 dilutions, Novus Biologicals, Inc).

Techniques: Sequencing, Clone Assay, Phospho-proteomics, Glycoproteomics, Binding Assay, Stable Transfection, Transfection, Cell Culture, Inhibition, cAMP Assay

Journal: Circulation Research

Article Title: Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells

doi: 10.1161/01.res.0000165800.81876.52

Figure Lengend Snippet: Figure 6. Effects of A1R antisense OGN and overexpression on the DNA synthesis of CASMCs. CASMCs were pretreated with vehicle (control), transfection reagent (Trans. Reagent), antisense oligonucleotides to porcine A1R (antisense OGN, 0.3 g), or the sense OGN (0.3 g) (A). Western blotting was performed to confirm the knockdown of endogenous A1R. Molecular weights of A1R and actin are 39 and 43 kDa, respectively. In parallel, pretreated CASMCs were stimulated with adenosine (100 mol/L), NECA (10 mol/L), CCPA (0.1 mol/L), or LPA (100 mol/L) for 48 hours, after which cellular DNA content was determined and the data were transformed to the percentage inhibition of the controls (B). In another experiment, CASMCs were transfected with vehicle (control), transfection reagent (Trans. Reagent), pCR3.1 containing the porcine A1R cDNA (Plasmid-A1R), or mock pCR3.1 (C). Western blotting was performed to confirm A1R overexpression. In parallel, the cellular DNA content was determined after stimulation of posttransfected CASMCs with adenosine (100 mol/L), NECA (10 mol/L), CCPA (0.1 mol/L), or LPA (100 mol/L) for 48 hours (D). Data shown are the meanSEM from four independent experiments performed in triplicate. *P0.01 relative to the respective control.

Article Snippet: Cells were lysed and lysates Western blotted for the A1R (rabbit polyclonal antibody, 1:1000 dilutions, Novus Biologicals, Inc).

Techniques: Over Expression, DNA Synthesis, Control, Transfection, Western Blot, Knockdown, Transformation Assay, Inhibition, Plasmid Preparation

Journal: Circulation Research

Article Title: Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells

doi: 10.1161/01.res.0000165800.81876.52

Figure Lengend Snippet: Figure 8. A1R mediate adenosine- and CCPA-induced DNA syn- thesis in an organ culture model of coronary disease. Artery rings of porcine conduit coronary arteries denuded of endotheli- um were stimulated with the indicated concentrations of adeno- sine, CCPA, PDGF, or the vehicle (control) for 24 hours in organ culture, after which the cellular DNA content was determined (A). Alternatively, artery rings were pretreated with DPCPX (0.1 mol/L, 40 minutes) and stimulated by adenosine (10 mol/L), CCPA (10 nmol/L), or PDGF (10 nmol/L), after which the cellular DNA content was determined (B). Data shown are the meanSEM from 4 independent experiments (4 pigs) performed in duplicate. *P0.05 relative to the respective control.

Article Snippet: Cells were lysed and lysates Western blotted for the A1R (rabbit polyclonal antibody, 1:1000 dilutions, Novus Biologicals, Inc).

Techniques: Organ Culture, Control