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Results: 11

1.
FIGURE 3

FIGURE 3. From: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation.

Adenosine has no effect on IFN-γ-induced STAT1 tyrosine phosphorylation in mouse macrophages. A, Whole-cell lysates were prepared and subjected to Western blot analysis. A representative immunoblot of phospho-STAT1 at Y701 is shown. M, media; A, adenosine; I, IFN-γ (IFNg); I+A, IFN-γ plus adenosine; n = 3. B, Densitometric analysis of Western blot. STAT1 Y701 bands from cells 10, 20, and 240 min post-treatment were normalized to total STAT1 and represented as fold change from untreated (M) cells. Dotted line, baseline control. Adenosine had no effect on STAT1 phosphotyrosine status in activated macrophages (from two-way ANOVA); n = 3.

Kimberly E. Barnholt, et al. J Immunol. ;183(10):6767-6777.
2.
FIGURE 2

FIGURE 2. From: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation.

Adenosine reduces IFN-γ-induced phosphorylation at the STAT1 S727 residue in mouse macrophages. A, Whole-cell lysates were prepared and subjected to Western blot analysis. A representative immunoblot of phospho-STAT1 at S727 is shown. M, medium; A, adenosine; I, IFN-γ (IFNg); I + A, IFN-γ plus adenosine; n = 3. B, Densitometric analysis of Western blot. STAT1 S727 bands from cells 10, 20, and 240 min posttreatment were normalized to total STAT1 and represented as fold change from untreated (M) cells. Dotted line, baseline control. Adenosine treatment reduced STAT1 S727 phosphorylation in activated macrophages compared with IFN-γ alone. *, p < 0.01 from two-way ANOVA; n = 3.

Kimberly E. Barnholt, et al. J Immunol. ;183(10):6767-6777.
3.
FIGURE 1

FIGURE 1. From: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation.

Adenosine attenuates IFN-γ-induced gene expression in a dose-dependent manner. RAW 264.7 macrophages were stimulated with IFN-γ (1000 U/ml) and various concentrations of adenosine (0 –1000 μM). Changes in expression of select genes were monitored by qRT-PCR and normalized to expression values of GAPDH. Results are expressed as fold change from the medium control samples. Adenosine treatment reduced expression of a STAT1-dependent transcription factor (A; IRF1), proinflammatory mediator (B; iNOS), and scavenger receptor (C; CD36) in a concentration-dependent manner. Statistics determined by one-way ANOVA. *, p < 0.05; **, p < 0.01; and ***, p ≤ 0.001 compared with cells treated with IFN-γ alone; n = 4.

Kimberly E. Barnholt, et al. J Immunol. ;183(10):6767-6777.
4.
FIGURE 9

FIGURE 9. From: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation.

Adenosine inhibits phosphorylation of STAT1 at S727 but not at Y701 in IFN-γ-treated human macrophages. Whole-cell lysates from THP-1 monocyte-derived macrophages were prepared and subjected to Western blot analysis. THP-1 macrophages were treated with medium (untreated control), IFN-γ (IFNg; 1000 U/ml), IFN-γ plus adenosine (Ado; 100 μM), or IFN-γ plus adenosine (Ado; 300 μM). Phosphorylated STAT1 bands were analyzed by densitometry, normalized to total STAT1, and represented as fold change from medium control. A, Representative immunoblot and cumulative densitometric analysis of phospho-STAT1 at S727. B, Representative immunoblot and cumulative densitometric analysis of phospho-STAT1 at Y701. Adenosine treatment attenuated the IFN-γ-induced phosphorylation of STAT1 at S727 in a dose-dependent manner. Adenosine had no effect on STAT1 Y701 band intensity in activated human macrophages. Statistics determined by one-way ANOVA on Ranks. *, p < 0.05 compared with control; †, p < 0.05 compared with cells treated with IFN-γ alone; n = 5.

Kimberly E. Barnholt, et al. J Immunol. ;183(10):6767-6777.
5.
FIGURE 4

FIGURE 4. From: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation.

Treatment of IFN-γ-stimulated RAW 264.7 macrophages with adenosine decreases STAT1-responsive promoter activity. Cells were transfected with a STAT1 homodimer reporter vector (GAS-Luc) mixed with a Renilla luciferase control. Twenty-four hours posttransfection, cells were treated with IFN-γ (IFNg) or IFN-γ plus adenosine (IFN-γ + Ado) for 10, 20, 60, 120, or 240 min. Values represent promoter activity expressed as relative luciferase activity after correction for Renilla luciferase activity and normalization to untreated cells. Adenosine treatment significantly reduced STAT1-responsive promoter activity in IFN-γ-treated macrophages starting at 60 min posttreatment. Statistics were performed on transformed data (log10), and significance was determined by two-way ANOVA. *, p ≤ 0.001 compared with cells treated with IFN-γ alone; n = 3.

Kimberly E. Barnholt, et al. J Immunol. ;183(10):6767-6777.
6.
FIGURE 7

FIGURE 7. From: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation.

A3 receptor signaling reduces IFN-γ-induced phosphorylation of STAT1 at S727 but not Y701. Whole-cell lysates from RAW 264.7 macrophages were prepared and subjected to Western blot analysis. Treatments include medium, IFN-γ (IFNg), IFN-γ plus aden-osine (Ado), or IFN-γ plus adenosine plus MRS 1191 (A3 receptor antagonist). Phosphorylated STAT1 bands were analyzed by densitometry, normalized to total STAT1, and represented as fold change from medium control. A, Representative immunoblot and cumulative densitometric analysis of phospho-STAT1 at S727. B, Representative immunoblot and cumulative densitometric analysis of phospho-STAT1 at Y701. Adenosine treatment attenuated the IFN-γ-induced phosphorylation of STAT1 at S727. Inhibition of A3 receptor signaling with MRS 1191 abrogated the suppressive effect of adenosine, suggesting a role for this receptor subtype in STAT1 modulation. Adenosine had no effect on STAT1 Y701 band intensity in activated macrophages. Statistics determined by one-way ANOVA on ranks. *, p < 0.05 compared with control; †, p < 0.05 compared with cells treated with IFN-γ alone; n = 4.

Kimberly E. Barnholt, et al. J Immunol. ;183(10):6767-6777.
7.
FIGURE 5

FIGURE 5. From: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation.

Adenosine treatment does not alter STAT1 DNA binding. Nuclear extract (12.5 μg) from each sample was used in a transcription factor assay to determine STAT1 DNA binding. The assay was performed in the absence or presence of competitor oligonucleotide (oligos; 20 pmol) containing a wild-type STAT1 consensus binding site. Nuclear extract from IFN-γ (IFNg)-stimulated RAW 264.7 macrophages (both with and without adenosine treatment) showed significantly higher STAT1 activation than did extract from untreated cells. *, p < 0.001 determined by one-way ANOVA. There was no difference in STAT1 DNA binding between cells stimulated with IFN-γ alone or those treated with IFN-γ plus adenosine at any time point. The addition of wild-type competitor oligo-nucleotides to extract significantly reduced STAT1 activation in all cells stimulated with IFN-γ (both with and without adenosine treatment) compared with cell extract alone. †, p < 0.001 determined by pairwise comparison). n = 4 for extract results; n = 3 for extract with wild-type competitor oligonucleotides.

Kimberly E. Barnholt, et al. J Immunol. ;183(10):6767-6777.
8.
FIGURE 8

FIGURE 8. From: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation.

A3 receptor stimulation is necessary for adenosine-mediated suppression of STAT1-dependent genes in activated macrophages. RAW 264.7 cells were treated for 4 h with IFN-γ (IFNg), IFN-γ plus Cl-IB-MECA, or IFN-γ plus Cl-IB-MECA plus MRS 1191. The selective A3 adenosine receptor agonist Cl-IB-MECA was added to cells 30 min before the IFN-γ challenge. MRS 1191, a selective antagonist of the A3 adenosine receptor, was added 20 min before Cl-IB-MECA and 50 min before IFN-γ treatment. Changes in IRF1 (A) and iNOS (B) gene expression were determined by qRT-PCR, normalized to GAPDH, and expressed as fold change from an untreated control. Cl-IB-MECA attenuated the IFN-γ-induced expression of IRF1 and iNOS. This inhibitory effect was reversed by MRS 1191, suggesting that the adenosine-mediated block in STAT1-dependent gene expression is due to A3 receptor specific stimulation. Statistics determined by one-way ANOVA. ***, p < 0.001 compared with cells treated with IFN-γ alone; n = 6.

Kimberly E. Barnholt, et al. J Immunol. ;183(10):6767-6777.
9.
FIGURE 11

FIGURE 11. From: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation.

Adenosine signaling has no effect on IFN-γ-induced STAT1 tyrosine phosphorylation in THP-1 cells. Whole-cell lysates from THP-1 monocyte-derived macrophages were prepared and subjected to Western blot analysis. Treatments included media, IFN-γ (IFNg), IFN-γ plus adenosine (Ado) (100 μM), or IFN-γ plus adenosine plus one of the selective adenosine receptor antagonists (10 μM): CPX, A1 receptor antagonist; SCH 58261 (SCH), A2A receptor antagonist; alloxazine (Allox), A2B receptor antagonist; MRS 1220 (MRS), human A3 receptor antagonist. A representative immunoblot and densitometric analysis of phospho-STAT1 at Y701 are shown. STAT1 Y701 bands were normalized to total STAT1 and represented as fold change from control medium. STAT1 phosphotyrosine status in IFN-γ-stimulated macrophages was unaffected by adenosine signaling through any of its receptor subtypes. Statistics determined by one-way ANOVA. ***, p < 0.001 compared with control; n = 3.

Kimberly E. Barnholt, et al. J Immunol. ;183(10):6767-6777.
10.
FIGURE 6

FIGURE 6. From: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation.

Selective stimulation of the A3 receptor reduces STAT1 transcriptional activity in IFN-γ-treated macrophages. RAW 264.7 cells were cotransfected with a STAT1 homodimer reporter vector (GAS-Luc) mixed with a Renilla luciferase control. Twenty-four hours posttransfection, cells were treated for 4 h with IFN-γ (IFNg) or IFN-γ plus one of the selective adenosine receptor agonists: A, CCPA, A1 receptor agonist; B, CGS21680 (CGS), A2A receptor agonist; C, NECA, nonselective A1 and A2 receptor agonist; D, Cl-IB-MECA, A3 receptor agonist. Agonists were added to cells 30 min before treatment with IFN-γ. Promoter activity values are expressed as relative luciferase units after correction for Renilla luciferase activity and normalization to untreated cells. Only treatment with the A3 receptor-specific agonist (Cl-IB-MECA) led to a significant decrease in STAT1-responsive promoter activity in IFN-γ-stimulated cells. Statistics determined by Student's t test. *, p < 0.01 compared with cells treated IFNwith IFN-γ alone. n = 7.

Kimberly E. Barnholt, et al. J Immunol. ;183(10):6767-6777.
11.
FIGURE 10

FIGURE 10. From: Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation.

Adenosine signaling through the A3 receptor suppresses IFN-γ-induced STAT1 S727 phosphorylation in human macrophages. Whole-cell lysates from THP-1 monocyte-derived macrophages were prepared and subjected to Western blot analysis. Treatments included medium, IFN-γ (IFNg), IFN-γ plus adenosine (100 μM), or IFN-γ plus aden-osine plus one of the selective adenosine receptor antagonists (10 μM): CPX, A1 receptor antagonist; SCH 58261 (SCH), A2A receptor antagonist; alloxazine; Allox; A2B receptor antagonist; MRS 1220 (MRS), human A3 receptor antagonist. A representative immunoblot and densitometric analysis of phospho-STAT1 at S727 are shown. STAT1 S727 bands were normalized to total STAT1 and represented as fold change from control (medium). Inhibition of A3 receptor signaling with MRS 1220 prevented the adenosine-mediated suppression of STAT1 S727 phosphorylation in IFN-γ-treated cells. This effect was not observed with inhibition of any other adenosine receptor subtype suggesting a unique role for the A3 receptor in modulating STAT1 activation. Statistics determined by one-way ANOVA. *, p < 0.05 compared with cells treated with IFN-γ alone; †, p < 0.05 compared with cells treated with IFN-γ plus adenosine plus MRS 1220; n = 3.

Kimberly E. Barnholt, et al. J Immunol. ;183(10):6767-6777.

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