Journal: Frontiers in Pharmacology

Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

doi: 10.3389/fphar.2021.619800

Figure Lengend Snippet: CAR T cell enhancement by BAY 60-6583 in CD133 CAR T cells seems not to be dependent on the adenosine A2b receptor. (A, B) Blocking of the adenosine A2b receptor cannot inhibit the enhanced antitumor activities induced by BAY 60-6583. In the anti-CD133 CAR T co-culture system (E:T ratio of 4:1), the adenosine A2b receptor was blocked by 1 μM PSB603 1 h before 10 μM BAY 60-6583 or 1 μM NECA was added. 24 h later, TNF-α production (A) and tumor lysis (B) were analyzed (C) The expression of the adenosine A2b receptor on conventional (WT) and A2b receptor knockout (4 + 6) anti-CD133 CAR T cells was determined by Western blot. Data from a representative experiment of n = 4 experiments (D, E) BAY 60-6583-induced enhancement of CAR T cell-mediated antitumor activities were not influenced by adenosine A2b receptor deficiency. Conventional and A2b receptor knockout anti-CD133 CAR T cells were co-cultured with U251-CD133OE luc cells at an E:T ratio of 4:1. After treatment with vehicle control or BAY 60-6583 for 24 h, cytokine secretion in the supernatant was detected (D) ; 48 h later, cytotoxicity was assessed (E) . * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by 1-way (A, B) or 2-way ANOVA (D, E) . Data are represented as the mean ± SD from a representative experiment of n = 3 experiments (A, B, D, E) .

Article Snippet: After blocking with 5% milk for 1 h, the membranes were incubated with anti-adenosine A2b receptor (AAR-003, alomone labs) or anti-GAPDH (sc-32233, Santa Cruz Biotechnology) antibodies at 4°C overnight.

Techniques: Blocking Assay, Co-Culture Assay, Lysis, Expressing, Knock-Out, Western Blot, Cell Culture

Journal: Disease Models & Mechanisms

Article Title: Adenosine and hyaluronan promote lung fibrosis and pulmonary hypertension in combined pulmonary fibrosis and emphysema

doi: 10.1242/dmm.038711

Figure Lengend Snippet: Cardiovascular physiology, metabolic profile, and hyaluronan synthesis and deposition in Ada −/− . (A-C) Right ventricular systolic pressure (RVSP; A), left ventricular systolic pressure (LVSP; B) and Fulton index ratio (C) determined by the measurement of right ventricle and left ventricle with septum from Ada + (white bars), Ada −/− (gray bars) and Ada −/− +4MU-treated (black bars) mice. (D-I) Transcript expression levels from lung tissue for Adora2b (D), Pparg (E), Sdha (F), Has1 (G), Has2 (H), Has3 (I) by RT-PCR from Ada + (white bars), Ada −/− (gray bars) and Ada −/− +4MU-treated (black bars) mice. (J) Representative dual-IHC staining in fibrotic areas of Ada −/− or Ada −/− +4MU treatment groups for αSMA (blue) and hyaluronan (magenta). Data are mean+s.e.m. ( n =5 per group). * P <0.05 (comparisons between Ada + and Ada −/− treatment groups), # P <0.05 (comparisons between Ada −/− and Ada −/− +4MU treatment groups); two-way ANOVA with the Benjamin, Krieger and Yekutieli post hoc test. Scale bars: 200 µm.

Article Snippet: The following antibodies were used: ADORA2B (rabbit polyclonal, Novus Biologicals, NBP2-41312; 1:2000); HAS3 (rabbit polyclonal, Proteintech, 15609-1-AP; 1:1000); GAPDH (mouse monoclonal, Invitrogen, AM4300; 1:500).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry

Journal: Disease Models & Mechanisms

Article Title: Adenosine and hyaluronan promote lung fibrosis and pulmonary hypertension in combined pulmonary fibrosis and emphysema

doi: 10.1242/dmm.038711

Figure Lengend Snippet: Response of macrophages to an ADORA2B agonist. (A-D) Transcript levels of Has1 (A), Has2 (B), Has3 (C) and IL6 (D) of MH-S cells exposed to DMSO (black bars), the ADORA2B antagonist GS-6201 (gray bars), the ADORA2B agonist BAY60-6583 (blue bars) or under the presence of both GS-6201 and BAY60-6583 (red bars). * P <0.05 (comparisons between DMSO and BAY60-6583 treatment groups). # P <0.05 (comparisons between BAY60-6583 and BAY60-6583+GS-6201 treatment groups); two-way ANOVA with the Benjamin, Krieger and Yekutieli post hoc test. Data are mean+s.e.m. ( n =5 for all groups).

Article Snippet: The following antibodies were used: ADORA2B (rabbit polyclonal, Novus Biologicals, NBP2-41312; 1:2000); HAS3 (rabbit polyclonal, Proteintech, 15609-1-AP; 1:1000); GAPDH (mouse monoclonal, Invitrogen, AM4300; 1:500).

Techniques:

Journal: Disease Models & Mechanisms

Article Title: Adenosine and hyaluronan promote lung fibrosis and pulmonary hypertension in combined pulmonary fibrosis and emphysema

doi: 10.1242/dmm.038711

Figure Lengend Snippet: The adenosinergic response in CPFE. (A-H) Lung transcript levels for CD39 (A), CD73 (B), ADA (C), ENT2 (D), ADORA1 (E), ADORA2A (F), ADORA2B (G) and ADORA3 (H) from upper lobes from normal lungs (white bars), upper lobes from CPFE lungs (light blue bars), lower lobes from normal lungs (gray bars) and lower lobes from CPFE lungs (dark blue bars). Data are mean+s.e.m. ( n =3 per group). * P <0.05 (comparisons between normal and CPFE groups within upper or lower lung lobes); unpaired two-tailed Student's t -test with a Welch correction.

Article Snippet: The following antibodies were used: ADORA2B (rabbit polyclonal, Novus Biologicals, NBP2-41312; 1:2000); HAS3 (rabbit polyclonal, Proteintech, 15609-1-AP; 1:1000); GAPDH (mouse monoclonal, Invitrogen, AM4300; 1:500).

Techniques: Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Adenosine and hyaluronan promote lung fibrosis and pulmonary hypertension in combined pulmonary fibrosis and emphysema

doi: 10.1242/dmm.038711

Figure Lengend Snippet: ADORA2B and HAS3 are elevated in CPFE. (A) Western blot for ADORA2B, HAS3 and GAPDH from upper and lower lobes from normal or CPFE lungs. (B,C) Densitometries for HAS3 (B) and ADORA2B (C) from upper lobes from normal lungs (white bars), upper lobes from CPFE lungs (light blue bars), lower lobes from normal lungs (gray bars) and lower lobes from CPFE lungs (dark blue bars). (D) Dual-IHC for αSMA (red/brown) and hyaluronan (blue) in normal (upper panels) or CPFE (lower panels) lungs. The red V denotes vessels and the green asterisks indicate myofibroblasts. (E) IHC for CD68 (blue) and HAS3 (red) from lower lobes from normal (left) and CPFE (right) lungs. Red arrowheads represent CD68-positive (HAS3-negative) signals. Black arrowheads indicate dual HAS3 and CD68 signals. (F) Double HAS3- and CD68-positive cells from ten micropictographs from the upper lobes from normal lungs (white bars), upper lobes from CPFE lungs (light blue bars), lower lobes from normal lungs (gray bars) and lower lobes from CPFE lungs (dark blue bars). Data are mean+s.e.m. ( n =3 per group). * P <0.05 (comparisons between normal and CPFE groups within upper or lower lung lobes); unpaired two-tailed Student's t -test with a Welch correction. Scale bars: 50 µm.

Article Snippet: The following antibodies were used: ADORA2B (rabbit polyclonal, Novus Biologicals, NBP2-41312; 1:2000); HAS3 (rabbit polyclonal, Proteintech, 15609-1-AP; 1:1000); GAPDH (mouse monoclonal, Invitrogen, AM4300; 1:500).

Techniques: Western Blot, Two Tailed Test

Journal: Hypertension

Article Title: Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D

doi: 10.1161/hypertensionaha.115.05912

Figure Lengend Snippet: Figure 1. Signaling schematic depicting our hypothesis of how adenosine regulates human coronary artery smooth muscle cell cell-cycle progression. Extracellular mitogens activate classical signal transduction pathways that ultimately phosphorylate (and thus activate) ERK1/2 and Akt. ERK1/2 is well known to increase expression of cyclin D (G1 phase cyclin). Phosphorylated Akt activates a signal transduction pathway that stabilizes S-phase kinase–associated protein-2 (Skp2), which is the F-box protein of SCFSkp2 ubiquitin ligase that polyubiquitinates p27Kip1 and thus accelerates the degradation of p27Kip1. Removal of p27Kip1 deinhibits cyclin D activity. Cyclin D activates cyclin-dependent kinases 4 and 6 to hyper-phosphorylate retinoblastoma protein (Rb), thus releasing the transcription factor elongation 2 factor (E2F) and allowing increased expression of G1/S and S phase cyclins. G1/S and S phase cyclins then drive the cell cycle forward to complete mitogenesis and cytokinesis (cell proliferation). Adenosine stimulates A2B receptors that are positively coupled to adenylyl cyclase, which increases the formation cAMP and activates protein kinase A. Protein kinase A decreases expression of Skp2 and inhibits phosphorylation of Akt, resulting in increased levels of p27Kip1 which reduce cyclin D activity. Protein kinase A also inhibits phosphorylation of ERK1/2, which reduces cyclin D expression. This protein kinase A (PKA)–induced signaling converges at cyclin D. This results in inhibition of Rb hyperphosphorylation, and hypophosphorylated Rb can now bind E2F and prevent this transcription factor from increasing the expression of G1/S and S phase cyclins.

Article Snippet: Primary Antibody (Source) Dilution of Primary Antibody (Time and Temperature of Incubation) Anti-Rb hypo/hyperphosphorylated (BD Biosciences) 1:1000 (overnight at 4°C) Anti-cyclin D1 (Upstate Biotechnology) 1:1000 (1 hour at room temperature; RT) Anti-β actin (Sigma) 1:10000 (40 min at RT) Anti-ERK1/2 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-ERK1/2 phosphorylated (Calbiochem) 1:1000 (1 hour at RT) Anti-Akt (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-Akt phosphorylated (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-cyclin A1 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-Skp2 (Cell Signalling) 1:1000 (2 hours at RT) Anti-p27 (Pharmigen) 1:250 (1 hour at RT) Anti-Adenosine Receptor A1 (Santa Cruz) 0.5-5 ug/ml (1 hour at RT) Anti A2A Adenosine receptor (Chemicon) 1:200 (1 hour at RT) 3 Anti A2B Adenosine receptor (Santa Cruz) 1:200 (1 hour at RT) Anti A3 Adenosine receptor (Santa Cruz) 1:200 (overnight at 4°C) Table S2: Effects of administration of 2-chloroadenosine (20 μmol/L in 25% pluronic acid) peri-arterially for 7 days on myointimal proliferation after balloon injury of carotid arteries of intact male Wistar-Kyoto rats.

Techniques: Transduction, Expressing, Ubiquitin Proteomics, Activity Assay, Phospho-proteomics, Inhibition

Journal: Hypertension

Article Title: Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D

doi: 10.1161/hypertensionaha.115.05912

Figure Lengend Snippet: Figure 2. Adenosine A2B receptor mediated inhibition of human coronary artery smooth muscle cell growth. A, Representative northern blots (upper) and western blots (lower) showing the presence of A1 and A2B receptors, with low expression of A2A receptors and minimal expression of A3 receptors. B, Concentration–response relationships for the inhibition of 3H-thymidine incorporation (DNA synthesis) by 2-chloroadenosine (Cl-Ad), 5′-N-methylcarboxamidoadenosine (MECA), 1-deoxy-1- [6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-d- ribofuranuronamide (IB-MECA), 5′-N-ethylcarboxamidoadenosine (NECA), N6-cyclopentyladenosine (CPA), and CGS21680 (CGS). C, The effects of Cl-Ad (1 μmol/L) on DNA synthesis in the presence and absence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; selective A1 antagonist, 100 nmol/L); SCH442416 (SCH; selective A2A antagonist, 100 nmol/L); VUF5574 (VUF; selective A3 antagonist, 100 nmol/L); and MRS1754 (MRS; selective A2B antagonist, 100 nmol/L). D, The inhibitory effects of Cl-Ad (100 nmol/L) on cell-cycle distribution. *P<0.05 vs no treatment; §P<0.05 significant reversal of Cl-Ad effects. Values represent mean±SEM from 3 separate experiments, each conducted in triplicates or quadruplicates.

Article Snippet: Primary Antibody (Source) Dilution of Primary Antibody (Time and Temperature of Incubation) Anti-Rb hypo/hyperphosphorylated (BD Biosciences) 1:1000 (overnight at 4°C) Anti-cyclin D1 (Upstate Biotechnology) 1:1000 (1 hour at room temperature; RT) Anti-β actin (Sigma) 1:10000 (40 min at RT) Anti-ERK1/2 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-ERK1/2 phosphorylated (Calbiochem) 1:1000 (1 hour at RT) Anti-Akt (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-Akt phosphorylated (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-cyclin A1 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-Skp2 (Cell Signalling) 1:1000 (2 hours at RT) Anti-p27 (Pharmigen) 1:250 (1 hour at RT) Anti-Adenosine Receptor A1 (Santa Cruz) 0.5-5 ug/ml (1 hour at RT) Anti A2A Adenosine receptor (Chemicon) 1:200 (1 hour at RT) 3 Anti A2B Adenosine receptor (Santa Cruz) 1:200 (1 hour at RT) Anti A3 Adenosine receptor (Santa Cruz) 1:200 (overnight at 4°C) Table S2: Effects of administration of 2-chloroadenosine (20 μmol/L in 25% pluronic acid) peri-arterially for 7 days on myointimal proliferation after balloon injury of carotid arteries of intact male Wistar-Kyoto rats.

Techniques: Inhibition, Northern Blot, Western Blot, Expressing, Concentration Assay, DNA Synthesis

Journal: Hypertension

Article Title: Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D

doi: 10.1161/hypertensionaha.115.05912

Figure Lengend Snippet: Figure 3. A, Bar graphs show the effects of 2-chloroadenosine (Cl-Ad; 1 μmol/L) and 5′-N-methylcarboxamidoadenosine (MECA; 1 μmol/L) on cell number in human coronary artery smooth muscle cells (HCASMCs). The inhibitory effects of Cl-Ad were reversed by MRS1754 (MRS; A2B receptor antagonist), but not by SCH442416 (SCH; A2A receptor antagonist), 8-cyclopentyl- 1,3-dipropylxanthine (DPCPX; A1 antagonist), or VUF5574 (VUF; A3 antagonist). Similar to Cl-Ad, the effects of MECA were blocked by MRS1754. *P<0.05 vs control; §significant reversal of the inhibitory effects. B, Bar graph demonstrates the effects of Cl-Ad (1 μmol/L) and MECA (1 μmol/L) on cell migration in HCASMCs. The inhibitory effects of Cl-Ad were mimicked by MECA, but not by N6-cyclopentyladenosine (CPA; A1 agonist), CGS21680 (CGS; A2A agonist), or 1-deoxy-1-[6-[[(3-iodophenyl) methyl]amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide (IB-MECA; IB-M: A3 adenosine receptor agonist). Moreover, the effects of Cl-Ad and MECA were reversed by MRS1754 (MRS; A2B receptor antagonist). *P<0.05 vs control; §significant reversal of the inhibitory effects. C, Effects of erythro-9-(2-hydroxy-3- nonyl)adenine (EHNA; 5 μmol/L; adenosine deaminase inhibitor) and 5-iodotubercidin (IDO; 0.1 μmol/L; adenosine kinase inhibitor) on cell number in HCASMCs. The inhibitory effects were significantly enhanced when the adenosine catabolism inhibitors EHNA+IDO were combined. Moreover, the effects of EHNA+IDO were reversed by MRS1754 (MRS; A2B receptor antagonist), but not by SCH442416 (SCH; A2A receptor antagonist), DPCPX (A1 antagonist), or VUF5574 (VUF; A3 antagonist), suggesting that endogenous adenosine inhibits HCASMC growth via A2B receptors. *P<0.05 vs control; §significant reversal of the inhibitory effects. Values represent mean±SEM from 3 separate experiments, each conducted in triplicates or quadruplicates.

Article Snippet: Primary Antibody (Source) Dilution of Primary Antibody (Time and Temperature of Incubation) Anti-Rb hypo/hyperphosphorylated (BD Biosciences) 1:1000 (overnight at 4°C) Anti-cyclin D1 (Upstate Biotechnology) 1:1000 (1 hour at room temperature; RT) Anti-β actin (Sigma) 1:10000 (40 min at RT) Anti-ERK1/2 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-ERK1/2 phosphorylated (Calbiochem) 1:1000 (1 hour at RT) Anti-Akt (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-Akt phosphorylated (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-cyclin A1 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-Skp2 (Cell Signalling) 1:1000 (2 hours at RT) Anti-p27 (Pharmigen) 1:250 (1 hour at RT) Anti-Adenosine Receptor A1 (Santa Cruz) 0.5-5 ug/ml (1 hour at RT) Anti A2A Adenosine receptor (Chemicon) 1:200 (1 hour at RT) 3 Anti A2B Adenosine receptor (Santa Cruz) 1:200 (1 hour at RT) Anti A3 Adenosine receptor (Santa Cruz) 1:200 (overnight at 4°C) Table S2: Effects of administration of 2-chloroadenosine (20 μmol/L in 25% pluronic acid) peri-arterially for 7 days on myointimal proliferation after balloon injury of carotid arteries of intact male Wistar-Kyoto rats.

Techniques: Control, Migration

Journal: Hypertension

Article Title: Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D

doi: 10.1161/hypertensionaha.115.05912

Figure Lengend Snippet: Figure 5. A (Top), Western blot depicting the downregulation of the expression of A2B receptors in human coronary artery smooth muscle cells (HCASMCs) by siRNA against A2B receptors. No treatment with siRNA (Con); treated with negative-control siRNA (si-Con); treated with siRNA against A2B receptor (siRNA). Bar graph for the western blot represents change in optical density ratio of A2B to β-actin. (Bottom), Depicts the effects of siRNA against A2B receptors on the stimulatory effects of 2-chloroadenosine (Cl-Ad; 1 μmol/L) on cAMP levels in HCASMCs. No treatment with siRNA (Control); treated with negative-control siRNA (siControl); treated with siRNA against A2B receptor (siRNA). *P<0.05 vs no Cl-Ad. B, Inhibitory effects of Cl-Ad, 5′-N-methylcarboxamidoadenosine (MECA), 5′-N- ethylcarboxamidoadenosine (NECA), N6-cyclopentyladenosine (CPA), 8-bromo-cAMP (cAMP), and erythro-9-(2-hydroxy-3- nonyl)adenine (EHNA; 10 μmol/L) plus 5-iodotubercidin (IDO; 0.1 μmol/L) on DNA synthesis in the absence and presence of A2B receptor siRNA in HCASMCs. No treatment with agonists (Control); treated with negative-control siRNA (si-Control); treated with siRNA against A2B receptor (A2B-siRNA)]. *P<0.05 vs no agonist; §significant reversal of the inhibitory effects. Values represent mean±SEM from 3 separate experiments, each conducted in triplicates.

Article Snippet: Primary Antibody (Source) Dilution of Primary Antibody (Time and Temperature of Incubation) Anti-Rb hypo/hyperphosphorylated (BD Biosciences) 1:1000 (overnight at 4°C) Anti-cyclin D1 (Upstate Biotechnology) 1:1000 (1 hour at room temperature; RT) Anti-β actin (Sigma) 1:10000 (40 min at RT) Anti-ERK1/2 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-ERK1/2 phosphorylated (Calbiochem) 1:1000 (1 hour at RT) Anti-Akt (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-Akt phosphorylated (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-cyclin A1 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-Skp2 (Cell Signalling) 1:1000 (2 hours at RT) Anti-p27 (Pharmigen) 1:250 (1 hour at RT) Anti-Adenosine Receptor A1 (Santa Cruz) 0.5-5 ug/ml (1 hour at RT) Anti A2A Adenosine receptor (Chemicon) 1:200 (1 hour at RT) 3 Anti A2B Adenosine receptor (Santa Cruz) 1:200 (1 hour at RT) Anti A3 Adenosine receptor (Santa Cruz) 1:200 (overnight at 4°C) Table S2: Effects of administration of 2-chloroadenosine (20 μmol/L in 25% pluronic acid) peri-arterially for 7 days on myointimal proliferation after balloon injury of carotid arteries of intact male Wistar-Kyoto rats.

Techniques: Western Blot, Expressing, Negative Control, Control, DNA Synthesis

Journal: Hypertension

Article Title: Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D

doi: 10.1161/hypertensionaha.115.05912

Figure Lengend Snippet: Figure 11. A, Concentration (0, 0.01, 0.1, 1 μmol/L)–response relationships for the inhibition of DNA synthesis (3H-thymidine incorporation) by 2-chloroadenosine (Cl-Ad) and adenosine (Ad) in human coronary artery smooth muscle cells (HCASMCs). B, The effects of 100 nmol/L of 2-chloroadenosine (Cl-Ad) and adenosine (Ad) on cell number in the presence and absence of MRS1754 (MRS; selective A2B antagonist, 100 nmol/L) or inhibitory effects of adenosine in presence or absence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; 1 μmol/L) plus 5-iodotubercidin (IDO; 0.1 μmol/L) and with or without MRS1754 (100nmol/L). C, The effects of adenosine on p27Kip1 and S-phase kinase–associated protein-2 (Skp2) expression in HCASMCs treated with or without adenosine (Ad; 100 nmol/L) for 48 hours. *P<0.05 vs no treatment; §P<0.05 significant reversal of inhibitory effects; #P<0.05 significant increase of inhibitory effects. Values represent mean±SEM from 3 separate experiments using separate HCASMCs, each conducted in triplicate. The optical density (OD) ratio in the bar graphs represents Skp2 or p27Kip1 to β-actin ratio.

Article Snippet: Primary Antibody (Source) Dilution of Primary Antibody (Time and Temperature of Incubation) Anti-Rb hypo/hyperphosphorylated (BD Biosciences) 1:1000 (overnight at 4°C) Anti-cyclin D1 (Upstate Biotechnology) 1:1000 (1 hour at room temperature; RT) Anti-β actin (Sigma) 1:10000 (40 min at RT) Anti-ERK1/2 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-ERK1/2 phosphorylated (Calbiochem) 1:1000 (1 hour at RT) Anti-Akt (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-Akt phosphorylated (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-cyclin A1 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-Skp2 (Cell Signalling) 1:1000 (2 hours at RT) Anti-p27 (Pharmigen) 1:250 (1 hour at RT) Anti-Adenosine Receptor A1 (Santa Cruz) 0.5-5 ug/ml (1 hour at RT) Anti A2A Adenosine receptor (Chemicon) 1:200 (1 hour at RT) 3 Anti A2B Adenosine receptor (Santa Cruz) 1:200 (1 hour at RT) Anti A3 Adenosine receptor (Santa Cruz) 1:200 (overnight at 4°C) Table S2: Effects of administration of 2-chloroadenosine (20 μmol/L in 25% pluronic acid) peri-arterially for 7 days on myointimal proliferation after balloon injury of carotid arteries of intact male Wistar-Kyoto rats.

Techniques: Concentration Assay, Inhibition, DNA Synthesis, Expressing