Silencing of <i>SDCBP</i> by siRNA inhibits uveal melanoma cell migration.

<p><b>A:</b> Western blot analysis of MEL 270 and 92.1 cell lines upon 72 hrs treatment with scrambled siRNA (C), and <i>SDCBP</i> targeting siRNA (Synt). <b>B:</b> wound-healing assay on MEL 270 and 92.1 cell lines treated with scrambled siRNA (C) or with <i>SDCBP</i> targeting siRNA (Synt). Mean of migration distance of MEL 270 cells (<b>C</b>) and 92.1 (<b>D</b>) treated with scrambled siRNA (black bars) or with <i>SDCBP</i> targeting siRNA (grey bars), P values are indicated.</p

Analysis of Mda-9/syntenin protein expression in uveal melanoma cell lines.

<p><b>A:</b> Immunostaining of fixed and permeabilized cell lines (left) and primary cultures (right). Original magnification 400×. The insets show the negative control performed by the use of non-immune rabbit Ig.</p

Silencing of mda-9/syntenin in 92.1 uveal melanoma cells inhibits in vitro invasion and HGF mediated signaling.

<p>A: Expression of c-MET in 92.1 cells detected by indirect immunofluorescence and flow-cytometry; c: negative control. B: Invasion of matrigel membranes by 92.1 cells towards different stimuli: medium with 10% serum (C), 50% conditioned medium from MG63 cell line (CM), 100 ng/ml recombinant HGF in 0.1% serum. C: Silencing of <i>SDCBP</i> (Synt-siRNA) in 92.1 cells inhibits their ability to invade matrigel membranes in response to conditioned medium from MG63 cell line (CM) or recombinant HGF (100 ng/ml). Data are presented as percentage of invading 92.1 cells treated with scrambled siRNA (C-siRNA). * p<0.04. D: Western blot showing inhibition of FAK, AKT and Src phosphorylation in <i>SDCBP</i>-silenced 92.1 cells compared to cells treated with scrambled siRNA. The same membrane was also stained for unphosphorylated FAK, AKT and Src , mda-9/syntenin and β-actin as protein loading control. E: Silencing of mda-9/syntenin in 92.1 uveal melanoma cells does not effect c-MET expression and p-MET phosphorylation. Western blot analysis of c-MET, p-MET, mda-9/syntenin and and β-actin as protein loading control in in 92.1 <i>SDCBP</i> silenced cells and control siRNA.</p

<i>SDCBP</i> mRNA is expressed in uveal melanoma cells.

<p><b>A:</b> Conventional RT-PCR analysis of <i>SDCBP</i> gene expression in cell lines derived from primary tumors (MEL 270 and 92.1), cell lines derived from metastatic lesions (OMM1 and OMM2.5) and from four primary cultures derived from patients' ocular tumors (1,2,3,4). The lane identified by “C-” indicates negative control in the absence of cDNA. <b>B:</b> qPCR analysis of <i>SDCBP</i> mRNA expression in uveal melanoma cell lines and primary cultures. Expression values are normalized on the mean of <i>GAPDH</i> gene expression.</p

Gene expression profile of primary uveal melanomas reveals high but heterogeneous expression of <i>SDCBP</i>.

<p><b>A:</b> Bars indicate intensitiy of <i>SDCBP</i> expression in 29 primary uveal melanoma analyzed by gene expression profiling in the present study. <b>B:</b> Heat map showing the expression levels of syndecan (<i>SDC</i>)-1, -2, 3 and -4 genes, <i>SDCBP</i> and syntenin-2 (<i>SDCBP2</i>). <b>C:</b> Comparrison of <i>SDCBP</i> expression in metastatic and non-metastatic patients (n = 29) in our cohort showed a trend to higher <i>SDCBP</i> expression in metastatic patients (p = 0.07). The same type of comparison performed on gene expression profile data from Onken et al. (D), between class1 (low-risk) and class 2 (high risk) patients (n = 27) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029989#pone.0029989-Onken1" target="_blank">[13]</a> and on gene expression profile data from Laurent et al. (E) between metastatic and non-metastatic patients (n = 63) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029989#pone.0029989-Laurent1" target="_blank">[35]</a> showed significantly elevated levels of <i>SDCBP</i> in high risk and metastic patients, respectively.</p

Immunohistochemical analysis of mda-9/syntenin in tissue sections of primary uveal melanomas shows correlation with metastatic progression.

<p><b>A:</b> mda-9/syntenin expression in a choroidal metastasis of colon adenocarcinoma <b>A′:</b> primary uveal melanoma stained by secondary antibody in the absence of anti-mda-9/syntenin antibody (negative control). <b>B, C, D:</b> representative primary uveal melanomas displaying low, medium or high levels of mda-9/syntenin expression, respectively (original magnification 400×). <b>E:</b> liver metastasis of uveal melanoma (original magnification 100×). <b>F:</b> the same section at higher magnification (200×). Arrows indicate single cells of UM positive for mda-9/syntenin, which infiltrate the mda-9/syntenin-negative liver parenchima. <b>G:</b> liver metastasis of uveal melanoma from a different patient. <b>H:</b> the same at higher magnification. <b>I:</b> Kaplan-Meier analysis of Mda-9/syntenin protein expression and disease-free survival in patients with primary tumors. Patients with low mda-9/syntenin expression (dark line) showed longer survival than patients with high expression (gray line) (P<0.014). Patients were stratified according to a combination of qualitative/semi-quantitative grading. Censored patients are indicated in each curve.</p

Clinicopathological characteristics of patient samples and expression of mda-9/syntenin by immunohistochemistry.

<p>Met: metastasis; Chr: chromosome; DFS: disease free survival. nd: not done; NP: not provided. In the eleventh column m: monosomy; d: disomy. In the twelft column p: polisomy; d: disomy. In the thirteenth column: previous patients treatment. In the fourteenth column: mda-9/syntenin expression level, L: low expression; H: high expression.</p

Overexpression of mda-9/syntenin in Mel 270 uveal melanoma cells increases HGF-mediated signaling and invasiveness.

<p>A: Expression of c-MET in Mel 270 cells detected by indirect immunofluorescence and flow-cytometry; c: negative control. B: Transfection of <i>SDCBP</i> (SDCBP+) in Mel 270 cells enhances their ability to invade matrigel membranes in response to recombinant HGF (100 ng/ml). Data are presented as number of invading cells transfected with mda-9/syntenin vector or empty vector (mock). *: statistically significant difference between HGF induced invasion of mda-9/syntenin and mock transfected cells, p<0.04. C: Western blot analysis of FAK, AKT and Src phosphorylation in <i>SDCBP</i>-transfected Mel 270 cells compared to mock-transfected cells. The same membrane was also stained for unphosphorylated FAK, AKT and Src, mda-9/syntenin and β-actin protein as loading control. The second lane was cropped and repositioned.</p

Mda-9/syntenin is expressed in the nucleus of uveal melanoma cells.

<p><b>A:</b> Confocal fluorescence microscopy shows nuclear and cytoplasmic localization of mda-9/syntenin in 92.1 cells (upper panels) and OMM2.5 cells (lower panels). An optical section with mda-9/syntenin staining (green) and propidium iodide (red) is shown. A merging image is shown in the bottom quadrants of each panel (original magnification 600×). <b>B:</b> Western blot analysis showing nuclear and cytoplasmic expression of mda-9/syntenin. HDAC1 and β-actin were used as loading controls for nuclear and cytoplasmic extracts, respectively.</p

Mda-9/syntenin expression in a pseudo-metastatic model of uveal melanoma obtained by injection of human 92.1 cells under the spleen capsule of NOG mice: mda-9/syntenin expression is higher in liver metastases than in spleen.

<p><b>A:</b> Immunohistochemistry of murine splenic uveal melanoma and liver metastases (Original Magnification 400×). Arrows indicate single cells of uveal melanoma strongly positive for mda-9/syntenin present in the spleen; arrowheads indicate mda-9/syntenin positive metastatic cells in the liver. <b>B:</b> Flow-cytometric analysis of intracellular mda-9/syntenin expression in permeabilized 92.1 cell derived from splenic tumor and liver metastases, C- is the negative control.</p