Resveratrol, UVB or both and HaCaT cells proliferation.

<p>Resveratrol (25 and 100 µM) was added to HaCaT cells for 2 hours (panel A) or 24 hours (panel B) and withdrawn prior to irradiation with UVB (10, 20, 40, and 100 mJ/cm²). Cell count was performed after 48 hours in culture in standard medium. Graphs report mean values ± standard deviation of three independent measurement for each experimental point. RSV: resveratrol; * p<0.05.</p

Multiple pathway analysis in HaCaT cells exposed to resveratrol, UVB or both: earlier time point (15 minutes).

<p>HaCaT cells were pretreated with 25 and 100 µM resveratrol for 2 and 24 hours prior irradiation with UVB (10, 40, and 100 mJ/cm²). Cell lysates were collected 15′ after UVB irradiation. Phosphorylated or not phosphorylated protein levels were analyzed by western blot. The figure shows representative blots analyzing the levels of the phosphorylated forms of ERK1/2, p38, p53, AKT, and S6, and the levels of BAX, and Bcl2.GAPDH was used as loading control.</p

Relative contribution of apoptosis and autophagy to resveratrol enhanced UVB-induced HaCaT cells death.

<p>(A) HaCaT cells were pretreated for 24 hours with 25 µM resveratrol followed by 2 hours in the presence or absence of pan caspase inhibitor Z-VAD (50 µM) prior to irradiation with UVB (30 mJ/cm²). Cell count was performed after additional 24 hours of culture in standard medium. Graphs report mean values ± standard deviation of three independent measurements for each experimental point. RSV: resveratrol; * p<0.05. (B) “Total” represents the difference of cell number between UVB-irradiated HaCaT cells and resveratrol pre-treated and UVB-irradiated HaCaT cells; “Apoptotic” represent the difference of cell number between resveratrol/Z-VAD pre-treated/UVB-irradiated HaCaT cells and resveratrol pre-treated and UVB-irradiated HaCaT cells; Both “Total” and “Apoptotic” have been reported as percentage of “Total”(100% and 45% respectively). “Non-apoptotic” (55%) represent the difference of the percentages between “Total” and “Apoptotic”.</p

Cleavage of caspase 8 and PARP in HaCaT cells exposed to resveratrol, UVB or both.

<p>HaCaT cells were treated with resveratrol (25 and 100 µM, 2 and 24 hours), UVB (10, 40, and 100 mJ/cm² alone or pretreated for 2 or 24 hours with resveratrol (25 and 100 µM) prior irradiation with UVB (10, 40, and 100 mJ/cm²). Lysates were collected 4 hours and 30′ after irradiation and resveratrol withdrawal and challenged with anti-caspase 8 and anti PARP antibodies recognizing the cleaved forms of the two proteins (panel A), or with anti-Beclin 1. Panel B reports the densitometric analysis of caspase 8 cleaved band and Beclin 1 at both 2 and 24 hours prior irradiation. The same lysates were challenged with anti γ-H2AX, recognizing the phosphorylated form of the histone (Panel D). GAPDH was used as loading control.</p

Analysis of effects of UVB irradiation on autophagosomes and lysosomes compartments and on microtubule cytoskeleton in HaCaT cells untreated or pretreated with resveratrol.

<p>(A) HaCaT cells, in control conditions (CTR) or upon different treatments (RSV, UVB, RSV/UVB) were stained with monodansylcadaverin (MDC, in green) or with lysotracker (lysot., in red) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080728#s2" target="_blank">materials and methods</a>. Serial confocal sections were collected. The 3D reconstruction (black and white panels) is shown. Bar, 6 µm. (B) HaCaT cells, grown on coverslips, were loaded with lysotracker (in red), fixed and stained with a specific antibody against tubulin and revealed by FITC-conjugated secondary antibodies (green). Nuclei were labeled with DAPI (blue). Serial confocal sections were collected. Bar, 8 µm. (C) Lc3-I to Lc3-II conversion analysis in HaCaT cells exposed to resveratrol, UVB or both. HaCaT cells were pretreated for 24 hours with 25 µM resveratrol in the presence or the absence of NH₄Cl (20 mM) and leupeptin (100 µM) and then irradiated with UVB (10, 40, and 100 mJ/cm²). Cell lysates were collected 15′ (panel A) or 4h30′ (panel B) after UVB irradiation and resveratrol withdrawal. A representative blot analyzing the levels of Lc3 I to Lc3 II conversion is shown. GAPDH was used as loading control.</p

ROS evaluation in HaCaT cells exposed to resveratrol, UVB or both.

<p>HaCaT cells were pretreated for 2(panel A), 8 hours (panel B) or 24 hours (panel C) with resveratrol (25 and 100 µM) and then irradiated with UVB (10, 40, and 100 mJ/cm²). The levels of ROS were estimated indirectly by measuring the fluorescence emitted by dichlorofluoresceine (DCF) formed in proportion to the intracellular ROS 30′ after UVB irradiation. Graphs report mean values ± standard deviations of three independent measurement for each experimental point. RSV: resveratrol; a.u.: arbitrary units; * p<0.05.</p

Growth curves of HaCaT cells exposed to resveratrol, UVB or both.

<p>Resveratrol (25 µM) was added to HaCaT cells for 24 hours and withdrawn prior to irradiation with UVB (10 or 20 mJ/cm²). Cells were counted after 48, and 120 hours in culture. <b>⧫</b> Untreated; ▪ RSV 25 µM; ▴UVB 10 mJ; Δ RSV + UVB 10 mJ; •UVB 20 mJ; o RSV + UVB 20 mJ.</p

(A) ROS production in HaCaT cells and HaCaT cells were pretreated for 24 hours with resveratrol (25 and 100 µM) prior to the addition of H₂O₂ (200, 400, and 800 µM).

<p>The levels of ROS were estimated indirectly by measuring the fluorescence emitted by dichlorofluoresceine (DCF) formed in proportion to the intracellular ROS 30′ after addition of H₂O₂. Graphs report mean values ± standard deviation of three independent measurement for each experimental point. * p<0,05 <b>(B) HaCaT cell proliferation after exposure to resveratrol, H₂O₂ or both.</b> Resveratrol (25 and 100 µM) was added to HaCaT cells for 24 hours and withdrawn prior addition of H₂O₂ (200, 400, and 800 µM) and cell number was determined after additional 48 hours in culture. Graphs report mean values ± the standard deviation of three independent measurements for each experimental point. * p<0.05.</p