Influence of LPS on cell viability.

<p>U937 cell were exposed to 10 µg/mL of LPS for 24 h and cell viability was assessed by MTT assay. Results are expressed in percentage of control (mean ± SEM of five independent experiments, performed in triplicate). *p<0.05 vs control cells (0 µg/mL of LPS); **p<0.01 vs control cells.</p

Effect of ASTA on expression and activity of HO-1.

<p>A) Western blot and quantitative analysis for HO-1 expression, actin being used as loading control; data shown are means ± SD, *<i>p</i><0.05 compared with control cells; ^§<i>p</i><0.05 compared with LPS or ASTA treated cells; data were from at least three independent experiments, each performed in triplicate. B) Mean HO activity (nmol/bilirubin/mg protein)/h ± SD, n = 3) detectable in homogenates obtained from U937 cells: untreated; treated with LPS, ASTA and LPS+ASTA. *<i>p</i><0.05 compared with control cells; ^§<i>p</i><0.05 compared with LPS or ASTA treated cells.</p

Effect of ASTA on expression of Nrf2.

<p>U937 cells were treated with LPS or ASTA at the doses indicated. Groups include the following: Nuclear, Nrf2 protein levels in nuclear extracts; Cytosolic, Nrf2 protein levels in cytosolic fractions; panels on left show results of Western blot as appropriate. Plot on right shows the results of quantitative analysis. *<i>p</i><0.05 compared with control cells; ^§<i>p</i><0.05 compared with LPS-treated cells.</p

The effect of HO-1 inhibition and ASTA treatment on cellular superoxide anion release.

<p>Values are means±SD of values from three experiments. The control value (no addition of ASTA) was set at 1. *p<0.05 vs control cells; ^§p<0.05 vs. LPS treated cells.</p

Effects of ASTA on viability of U937 cells.

<p>Cells were incubated with or without ASTA (10 µM) for 1 h, and then stimulated with LPS (10 µg/ml) for another 24 h. Cell viability was determined as described in the Materials and Methods section. Data are the mean values of three experiments ± SEM. *p<0.05 vs. control cells; ^§p<0.05 vs. LPS or ASTA treated cells.</p

Effect of ASTA treatment on superoxide anion generation assessed by estimating reduced nitrobluetetrazolium (NBT) inU937 cells.

<p>O₂⁻ production increased significantly in U937 cells stimulated with LPS. ASTA blocked LPS-induced O₂⁻ production. Values are means±SD of values from three experiments. The control value (no addition of ASTA) was set at 1. *p<0.05 vs control cells; ^§p<0.05 vs. LPS or ASTA treated cells.</p

mTOR Activation by PI3K/Akt and ERK Signaling in Short ELF-EMF Exposed Human Keratinocytes

<div><p>Several reports suggest that ELF-EMF exposures interact with biological processes including promotion of cell proliferation. However, the molecular mechanisms by which ELF-EMF controls cell growth are not completely understood. The present study aimed to investigate the effect of ELF-EMF on keratinocytes proliferation and molecular mechanisms involved. Effect of ELF-EMF (50 Hz, 1 mT) on HaCaT cell cycle and cells growth and viability was monitored by FACS analysis and BrdU assay. Gene expression profile by microarray and qRT-PCR validation was performed in HaCaT cells exposed or not to ELF-EMF. mTOR, Akt and MAPKs expressions were evaluated by Western blot analysis. In HaCaT cells, short ELF-EMF exposure modulates distinct patterns of gene expression involved in cell proliferation and in the cell cycle. mTOR activation resulted the main molecular target of ELF-EMF on HaCaT cells. Our data showed the increase of the canonical pathway of mTOR regulation (PI3K/Akt) and activation of ERK signaling pathways. Our results indicate that ELF-EMF selectively modulated the expression of multiple genes related to pivotal biological processes and functions that play a key role in physio-pathological mechanisms such as wound healing.</p></div

Effects of pharmacological inhibitors on HaCaT cells proliferation.

<p>Proliferative rate of HaCaT cells assed by BrdU incorporation assay, after 24 h of incubation in non-exposed and exposed to ELF-EMF only for the first hour. Cells were pre-treated or not with selective inhibitor of ERK kinase activity (PD980559, 1 μM), PI3K (Ly294002, 1 μM), JNK Inhibitor II (20 μM) for 30 min. Each bar represents the mean±SD of three independent experiments performed in triplicate.</p

Effect of ELF-EMF exposure on AKT and MAPK activity.

<p>A. Representative image of immunoblotting for p-ERK, p-JNK, p-p38 and p-AKT of gels using three separate pools of protein extracted from HaCaT cells time-course exposed to ELF-EMF. B. Averaged band density of p-ERK, p-JNK, p-p38-immunoreactive and p-AKT is expressed as relative expression in both exposed and non-exposed to ELF-EMF (mean±SD; ^#<i>p<0</i>.<i>05 vs</i> time related non-exposed cells).</p

Cell cycle analysis.

<p>Flow cytometry cell cycle analysis of HaCaT cells exposed to ELF-EMF (1 mT, 50 Hz) for 1 and 24 h, using PI (40 μg/ml) as probe. Percentage of fluorescent cells for G0/G1, S and G2/M phases were reported in the bottom panel. Mean±SD of three analyses is reported. *<i>p<0</i>.<i>05 vs</i> non-exposed cells. </p