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1.
Figure 5

Figure 5. From: Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia.

Cytokine-induced Stat1 activation does not persist during subsequent TLR2 stimulation. A) Phosphorylated and total Stat1 cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 30 min. B). Phosphorylated and total Stat1 cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were first treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 24 hours, followed by incubation without or with Pam3CSK4 (25 mg/ml) for 30 min.

Audra A Winder, et al. Respir Res. 2009;10(1):96-96.
2.
Figure 3

Figure 3. From: Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia.

A) Effects of cytokines and dexamethasone (Dex) on TLR2-mediated IL-8 production. Epithelia were pre-treated overnight (18 hr) with cytokines (TNF-α and IFN-γ), dexamethasone, or a combination of cytokines plus dexamethasone, then exposed to Pam3CSK4 (25 μg/ml) (black), or control serum-free media (grey) for a period of 24 hours. Data represent the mean ± SEM from 5 donor samples. *P < 0.05. B) Polarity of TLR2 responses in airway epithelia. Epithelia were pre-treated overnight (18 hr) with cytokines (TNF-α and IFN-γ), then exposed to Pam3CSK4 (25 μg/ml), applied to either the apical (white) or basolateral (BL, black) surface, or control serum-free media (shown in grey) for a period of 24 hours. Data represent the mean ± SEM from 5 donor samples. *P < 0.05.

Audra A Winder, et al. Respir Res. 2009;10(1):96-96.
3.
Figure 7

Figure 7. From: Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia.

A) Effects of cytokines and dexamethasone (Dex) on TLR2-mediated HBD-2 expression. Epithelia were pre-treated overnight (18 hr) with cytokines (TNF-α and IFN-γ), dexamethasone, or a combination of cytokines plus dexamethasone, then exposed to Pam3CSK4 (25 μg/ml) (black), or control serum-free media (grey), for a 24-hour period. HBD-2 mRNA abundance determined by quantitative PCR. Data represent mean ± SEM from 5 donor samples. *P < 0.05, **P < 0.01. B) Effects of cytokines and dexamethasone (Dex) on TLR2-mediated CCL20 release. Primary cultures were pre-treated overnight with cytokines (TNF-α and IFN-γ), dexamethasone, or combination of cytokines plus dexamethasone, then exposed to Pam3CSK4 (25 μg/ml) (black), or control serum-free media (grey) for a period of 24 hours. Data represent mean ± SEM from 5 donor samples. *P < 0.05.

Audra A Winder, et al. Respir Res. 2009;10(1):96-96.
4.
Figure 8

Figure 8. From: Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia.

TLR2-mediated ASL antimicrobial activity. Epithelia were pre-treated overnight with cytokines (TNF-α and IFN-γ), dexamethasone, or a combination of cytokines plus dexamethasone, then exposed to Pam3CSK4 (25 μg/ml) for 24 hours. Apical washings were obtained, and modified radial diffusion assays performed. Data represent fold change from control, TLR2-agonist exposed condition for the test organisms: Listeria monocytogenes (grey), Escherichia coli DH5α (white), and Pseudomonas aeruginosa PA01 (black). n = 3 donor samples. **P < 0.01.

Audra A Winder, et al. Respir Res. 2009;10(1):96-96.
5.
Figure 2

Figure 2. From: Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia.

A) Effects of cytokines and dexamethasone (Dex) on TLR2 mRNA expression. Cells were treated overnight with cytokines (TNF-α (100 ng/ml) and IFN-γ (100 ng/ml)), dexamethasone (1 μM), or combination of cytokines plus dexamethasone. Quantitative RT-PCR results are represented as fold change from the control (untreated) condition. Data are presented as the mean ± SEM of experiments on 6 donor samples. *P < 0.05, **P < 0.01. B) Effects of cytokines and dexamethasone (Dex) on TLR1 and TLR6 mRNA expression. Epithelia were treated as described in Figure 2A. Results of quantitative RT-PCR presented as fold change from control (untreated) condition for TLR1 mRNA (grey) and TLR6 mRNA (black). Data are presented as mean ± SEM from 4 donor samples. *P < 0.05.

Audra A Winder, et al. Respir Res. 2009;10(1):96-96.
6.
Figure 1

Figure 1. From: Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia.

Heatmap of microarray expression profiling in human airway epithelia following 24 hour stimulation with cytokines (IL-1β (100 ng/ml), TNF-α (100 ng/ml), and IFN-γ (100 ng/ml)) or control. Represented are expression profiles for microbial pattern recognition proteins including TLRs 1-10, NOD1, NOD2, and the PGRP family members. Also included are the IL-1 receptor variants 1 and 2 and proteins involved in endotoxin sensing (LBP, CD14, and MD-2). Dark blue signifies lowest expression, and dark red denotes highest expression levels. Asterisks indicate genes with significant cytokine induced increases in expression.

Audra A Winder, et al. Respir Res. 2009;10(1):96-96.
7.
Figure 6

Figure 6. From: Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia.

Cytokine-induced p38 MAP kinase activation does not augment subsequent TLR2 stimulation. A) Phosphorylated and total p38 cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 30 min. B) Phosphorylated and total p38, and heat shock protein-90 (HSP90) cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were first treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 24 hours, followed by incubation without or with Pam3CSK4 (25 mg/ml) for 30 min. C) Phosphorylated p38 and HSP90 protein levels in the experiment outlined in B were quantified using band densitometry of immunoblot analyses results with inclusion of samples from three individuals. Values were calculated as phosphorylated p38/HSP90, were normalized to the untreated control value for each individual, and are expressed as mean fold change in phosphorylated p38 ± S.D. (n = 3 in each group).

Audra A Winder, et al. Respir Res. 2009;10(1):96-96.
8.
Figure 4

Figure 4. From: Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia.

Cytokine-induced NF-κB activation is minimally augmented during subsequent TLR2 stimulation. A) IκBα and β-actin cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 30 min. B) IκBα and β-actin cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were first treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 24 hours, followed by incubation without or with Pam3CSK4 (25 mg/ml) for 30 min. C) IκBα and β-actin protein levels in the experiment outlined in B were quantified using band densitometry of immunoblot analyses results with inclusion of samples from three individuals. Values were calculated as IκBα/β-actin, were normalized to the untreated control value for each individual, and are expressed as mean fold change in IκBα ± S.D. (n = 3 in each group). D) NF-κB-dependent gene activation was assessed using luciferase activity assays of extracts from human airway epithelia that were initially infected with an adenoviral vector expressing a luciferase gene driven by four tandem NF-κB sites. Cells were then treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 24 hours, followed by incubation with Pam3CSK4 (25 mg/ml) for 24 hours. Values are expressed as mean ± S.D. (n = 2-3 samples from 4 individuals in each group), and a significant difference from the untreated control is indicated by an asterisk.

Audra A Winder, et al. Respir Res. 2009;10(1):96-96.

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