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Figure 6 | Respiratory Research

Figure 6

From: Epithelial expression of TLR4 is modulated in COPD and by steroids, salmeterol and cigarette smoke

Figure 6

Salmeterol reverses the inhibitory effect of dexamethasone on TLR4 membrane protein expression despite downregulation of mRNA. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in serum free medium and were left untreated or incubated with 10-6 M dexamethasone (Dex), 10-7M salmeterol (Sal) or both drugs in combination (Sal + Dex) for 16 hours. Numbers indicate Molar doses of drug. Following treatment, total RNA or membrane protein was extracted for PCR and Western blot analysis. For IL-8 expression analysis, cells were further stimulated with LPS 10 μg/ml × 24 hours. A. Real-time PCR analysis of TLR4 mRNA expression as a factor of β-actin expression. TLR4 expression was given an arbitrary value of 1 in control cells. Data are expressed as mean +/- S.E. and are obtained from three experiments. (* P = 0.05 compared to control). B. Western blot analysis of membrane extracts (10 μg) from A549 cells probed with an anti-TLR4. Densitometry was performed and corrected for corresponding ponsceau staining density. Data are expressed as mean +/- S.E. and are obtained from three experiments. (* P = 0.05 compared to control). C. Levels of IL-8 in supernatants were measured by ELISA and values are expressed as fold change compared to unstimulated control. Assays were performed in duplicate a minimum of three times. Values are expressed as mean +/- S.E. (n = 3). (** signifies P ≤ 0.005 of observed effect vs. LPS alone; † signifies P ≤ 0.05 of observed effect vs. LPS + Dex).

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