Abrogation of both constitutive and resveratrol-dependent EGFR and ERK phosphorylation by specific inhibitor of EGFR kinase.

<p>(<b>A</b>) Western blot analysis of whole-cell lysates. Keratinocytes were incubated with 2 µM PD168393 (PD16) for 30 minutes prior to addition of 50 µM resveratrol (Resv) for the indicated time-points. (<b>B</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 and ^§<i>P</i><0.01 <i>versus</i> untreated controls for each time-point. (<b>C</b>) Western blot analysis of whole-cell lysates. Keratinocytes were incubated with 2 µM PD16 prior to addition of 50 µM Rv for 1 h. Subsequently, cells were further treated for 12 h with 50 ng/ml TNFα. (<b>D</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 <i>versus</i> controls treated with TNFα only. Data are representative of three independent experiments.</p

Resveratrol did not protect phosphorylated EGFR from endogenous phosphatases, led to EGFR accumulation in the keratinocyte membranes, and induced its cytosolic degradation.

<p>Western blot analysis of the plasma membrane fraction, where cadherin was used as a loading control (<b>A</b>) and of the cytosolic fraction (<b>C</b>). After 24h treatment with resveratrol (Resv), keratinocytes were stimulated with TGFα (50 ng/ml) for 10 minutes. Then, 2 µM PD168393 (PD16) were added to the medium for further 10 minutes. (<b>B</b>, <b>D</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 and ^§<i>P</i><0.01 <i>versus</i> untreated controls.</p

Effects of resveratrol and verbascoside on the TNFα−induced phosphorylation of EGFR, ERK, and the NFκB subunit p65.

<p>(<b>A</b>) Western blot analysis performed in whole-cell lysates of human keratinocytes. Following 1 h pre-incubation with 50 µM polyphenol, cells were treated for further 12 h with TNFα (50 ng/ml). Actin was used as a loading control. (<b>B</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 <i>versus</i> untreated controls (0 h time-point); ^§<i>P</i><0.05 versus TNFα-treated conditions. (<b>C</b>) Binding activity of nuclear cell lysates to NFκB-specific or AP-1-specific DNA consensus sequences. *<i>P</i><0.05 <i>versus</i> untreated controls; ^§<i>P</i><0.05 versus TNFα-treated conditions. Data are representative of three independent experiments.</p

Effects of resveratrol and verbascoside on the phosphorylation status of EGFR, ERK1/2, and the NFκB subunit p65.

<p>(<b>A</b>) Western blot analysis performed in whole-cell lysates of human keratinocytes treated with 50 µM of the indicated polyphenol. Actin was used as a loading control. (<b>B, C, D</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 <i>versus</i> untreated controls (0 h time-point). (<b>E</b>) Western blot analysis of EGFR and ERK phosphorylation in whole-cell lysates of human keratinocytes treated with 10 and 50 µM Resv for 6 h. (<b>F</b>) Quantification of Western blot bands by densitometry. *<i>P</i><0.05 <i>versus</i> controls without Resv (0 µM). ^§<i>P</i><0.05 <i>versus</i> 10 µM Resv (0 µM). Data are representative of three independent experiments.</p

Resveratrol promoted nuclear accumulation of phosphorylated and non-phosphorylated EGFR alone and in association with TGFα.

<p>(<b>A</b>) Western blot analysis of nuclear levels of phosphorylated (nP-EGFR) and non-phosphorylated (nEGFR), and (<b>B</b>) its densitometric quantification. *<i>P</i><0.05 and ^§<i>P</i><0.01 <i>versus</i> untreated controls.</p

Effects of resveratrol on IL-8 expression are sensible to specific inhibitor of EGFR kinase.

<p>(<b>A</b>) Quantitative real-time RT-PCR measurement of <i>IL-8</i> mRNA. Cells were treated with 2 µM PD168393 (PD16) for 30 min, and then exposed to 50 µM resveratrol (Resv) for 1 h. Cells were cultivated for further 12 h, with or without TNFα (50 ng/ml). *<i>P</i><0.05 <i>versus</i> untreated controls; ^§<i>P</i><0.01 <i>versus</i> TNFα-treated conditions. (<b>B</b>) ELISA of IL-8 protein accumulation in the supernatants of human keratinocytes. Cells were treated with 2 µM PD16 for 30 min, and then, exposed to 50 µM Resv for 1 h. Cells were cultivated for further 24 h, with or without TNFα (100 ng/ml). Data are representative of three independent experiments.</p