Inhibitory effect of gliadin peptide 10-mer on IRAK1 phosphorylation and NF-kB activation.

<p>CACO-2/TC7 cells, either unstimulated or stimulated with PT-Gly (1mg/ml), p10-mer (50 µg/ml), p10-mer (50 µg/ml) + PT-Gly (1mg/ml), were analyzed by Western blot for IRAK1 phosphorylation and NF-kB activation. (<b>A</b>) Phosphorylated levels of IRAK1 (p-IRAK1) were analyzed in whole cell extracts by Western blot with anti-phospho-IRAK1 antibodies; for control, the blotted membranes were stripped and reprobed with anti-IRAK-1 antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. (<b>B</b>) NF-kB activation was analyzed in whole cell extracts by Western blot with anti-phospho-NF-kB p65 Ser antibodies; for control, the blotted membranes were stripped and reprobed with anti-NF-kB p65 antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. Densitometric analysis was performed using ImageJ version 1.46 software and peaks were reproduced by reading the Western Blot bands. One example representative of 3 experiments.</p

Effect of gliadin peptide 10-mer on p31–43 activity and entrance in CACO-2 cells.

<p>(<b>A</b>) CACO-2/TC7 cells, either unstimulated or stimulated with p31–43 (50 µg/ml), p10-mer (50 µg/ml) + p31–43 (50 µg/ml), were analyzed by Western blot for ERK phosphorylation and NF-kB activation. (<b>i</b>) Phosphorylated levels of ERK were analyzed in whole cell extracts by Western blot with anti-phospho-ERK1/2 antibodies; for control, the blotted membranes were stripped and reprobed with anti-ERK1/2 antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. (<b>ii</b>) NF-kB activation was analyzed in whole cell extracts by Western blot with anti-phospho-NF-kB p65 Ser antibodies; for control, the blotted membranes were stripped and reprobed with anti-NF-kB p65 antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. Densitometric analysis was performed using ImageJ version 1.46 software, peaks were reproduced by reading the Western Blot bands. One example representative of 3 experiments. (<b>B</b>) CACO-2/TC7 cells, either unstimulated or stimulated with biotinylated p31–43 (50 µg/ml), p10-mer (50 µg/ml) + biotinylated p31–43 (50 µg/ml), were immunostained with streptavidin-AlexaFluor and analyzed by a BD FACSCalibur flow cytometer. (<b>C</b>) CACO-2/TC7 cells, either unstimulated or stimulated with biotinylated p31–43 (50 µg/ml), p10-mer (50 µg/ml) + biotinylated p31–43 (50 µg/ml), were immunostained with streptavidin-AlexaFluor. The images were acquired using an Olympus U RFL fluorescence microscope.</p

Inhibitory effect of gliadin peptide 10-mer on ERK and p38 MAPK phosphorylation.

<p>CACO-2/TC7 cells either unstimulated or stimulated with PT-Gly (1mg/ml), p10-mer (50 µg/ml), p10-mer (50 µg/ml) + PT-Gly (1mg/ml), were analyzed by Western blot for ERK and p38 phosphorylation. Phosphorylated levels of ERK were analyzed in whole cell extracts by Western blot with anti-phospho-ERK1/2 antibodies; for control, the blotted membranes were stripped and reprobed with anti-ERK1/2 antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. (<b>B</b>) Phosphorylated levels of p38 MAPK were analyzed in whole cell extracts by Western blot with anti-phospho-p38 MAPK antibodies; for control, the blotted membranes were stripped and reprobed with anti-p38 MAPK antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. Densitometric analysis was performed using ImageJ version 1.46 software and peaks were reproduced by reading the Western Blot bands. One example representative of 3 experiments.</p

Inhibitory effect of gliadin peptide 10-mer on cytokine production.

<p>CACO-2/TC7 cells either unstimulated or stimulated with PT-Gly (1mg/ml), p10-mer (50 µg/ml), p10-mer (50 µg/ml) + PT-Gly (1mg/ml) were analyzed for cytokine production. Cell supernatants were analyzed for pro-inflammatory cytokines IL-6 (<b>A</b>) and IL-8 (<b>B</b>) release by an ELISA kit. Statistical analysis: *P<0.01 <i>versus</i> control; §P<0.05 <i>versus</i> PT-Gly. Results are expressed as mean ± SD; n = 3.</p

Effects of pro-proliferative stimuli on LNCaP cells.

<p>A) LNCaP proliferation induced by treatment with CCL5 and/or IL-6. ³H-TdR uptake of cells after 24 h of culture with CCL5 (grey bars), 72 h of culture with IL-6 (hatched bars) or 24 h of culture with combinations of CCL5 and IL-6 (crossed bars) at the indicated concentrations. B) ³H-TdR uptake of cells after 24 h culture with 5 ng/ml of CCL5 in the presence or in the absence of anti-CCR5 or 7E11c antibodies at the indicated concentrations; C) Western blot analysis of cell extracts (20 µg/lane) of LNCaP cells treated with CCL5 (5 ng/ml) and probed with anti-STAT 5 or with anti-cyclin D1 antibodies. Equal protein loading in all the lanes is confirmed by not specific bands shown in the middle gel. Arrows indicate phosphorylated STAT5 proteins. Western blotting results are representative of two independent experiments. D) ³H-TdR uptake of cells 72 h after binding or cross-linking of 7E11c or anti-PSMA antibodies. Values represent the mean±SEM of three independent experiments performed in triplicates. Stars on the bars indicate statistical significance as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004608#pone-0004608-g004" target="_blank">Fig. 4</a>.</p

Reactivity of anti-PSMA mAb.

<p>Panel A: representative flow cytometry of LNCaP cells stained with anti-PSMA or with J591 mAb. Controls are represented by cells goat anti-mouse-FITC IgG only. Panel B: flow cytometry of LNCaP cells incubated with biotinylated J591 mAb (B-J591) with or without pretreatment (45 min on ice) with anti-PSMA at the indicated concentrations or with non-biotinylated J591 mAb as control. Antibody binding was revealed by FITC-Avidin (F-Avidin). The percentage of positive cells and the Mean Fluorescence Intensity observed under the different conditions are representative of two independent experiments performed with independent cultures. Panel C: Western blot analysis of LNCaP cell lysates immuneprecipitated with the indicated mAbs, and then probed with the anti-PSMA mAb followed by a goat anti-mouse antiserum labelled with horseradish peroxidase. The apparent molecular weight of PSMA band is indicated by the arrow.</p

Kinetics of ERK1/2, p38, RAC1 and RAS activation in LNCaP cells following cross-linking of anti-PSMA, anti-VCAM-1 or 7E11c mAb^a).

a)<p>The 7E11c mAb recognizes an intracellular PSMA epitope and is therefore used as a further control of the experiment described.</p>b)<p>Results of gel densitometry are expressed as the ratio±SEM of activated/total protein as evaluated by measuring the respective areas.</p>c)<p>Statistical significance is reported with respect to the “No treatment” data.</p>d)<p>ns = not significant.</p>e)<p>nd = not determined.</p

Kinetics of ERK1/21/2, p38, RAC 1 and RAS phosphorylation induced by PSMA cross-linking.

<p>LNCaP cells were left untreated or subjected to PSMA cross-linking for the indicated times at 37°C. An anti-VCAM-1 mAb was used as the isotype matched control. Panels A and B: p38 and ERK1/2 activation was assessed in crude lysates. Equal amounts (20 µg) of total proteins were boiled in sample buffer and separated by SDS-PAGE. Following immunoblotting with an anti-phospho-p38 or anti-phospho-ERK1/2 mAb, immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibody and the ECL system. Panels C and D: RAC1 and RAS activation was evaluated by a specific assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004608#s2" target="_blank">Material and Methods</a> section. Bound active GTP-RAC and GTP-RAS molecules were analyzed by Western blotting using an anti-RAC1 or anti-Ras mAb and visualized as above. Total amount of ERK1/2, p38, RAC1 and RAS in crude lysates are shown as loading control at the bottom of each gel. Results are representative of one of three independent experiments.</p

Functional Cross Talk between CXCR4 and PDGFR on Glioblastoma Cells Is Essential for Migration

<div><p>Glioblastoma (GBM) is the most common and aggressive form of brain tumor, characterized by high migratory behavior and infiltration in brain parenchyma which render classic therapeutic approach ineffective. The migratory behaviour of GBM cells could be conditioned by a number of tissue- and glioma-derived cytokines and growth factors. Although the pro-migratory action of CXCL12 on GBM cells in vitro and in vivo is recognized, the molecular mechanisms involved are not clearly identified. In fact the signaling pathways involved in the pro-migratory action of CXCL12 may differ in individual glioblastoma and integrate with those resulting from abnormal expression and activation of growth factor receptors. In this study we investigated whether some of the receptor tyrosine kinases commonly expressed in GBM cells could cooperate with CXCL12/CXCR4 in their migratory behavior. Our results show a functional cross-talk between CXCR4 and PDGFR which appears to be essential for GBM chemotaxis.</p> </div

Inducing activity of PSMA cross-linking on IL-6 and CCL5 expression and role of PSMA-activated p38 and ERK1/2 .

<p>IL-6 (panel A) and CCL5 (panel B): production by LNCaP cells left untreated (open bars), stimulated by cross-linking via anti PSMA mAb (filled bars) or anti-VCAM-1 mAb (dashed bars) in the presence or in the absence of single or combined inhibitors (SB and PD) at the indicated doses (SB50 = 50 µg/ml, SB25 = 25 µg/ml; PD50 = 50 µg/ml, PD25 = 25 µg/ml; SB/PD25 = combinations of SB+PD at 25 µg/ml each). Control and treated cell cultures were washed, cultured for further 24 h before collecting cells and cell-free supernatants. IL-6 and CCL5 accumulation in each supernatant was measured by ELISA and expressed as pg/mg of cell lysates/24 h. Inhibitors were added to the cells 6 h prior to stimulation and maintained throughout the duration of the experiment. Values represent the mean±SEM of results of four experiments performed in duplicates. Stars on top of the bars indicate statistical significance as follows: p<0.001, ***; p<0.01, **; p<0.05, *.</p