Additional file 1: of IL-27 mediates HLA class I up-regulation, which can be inhibited by the IL-6 pathway, in HLA-deficient Small Cell Lung Cancer cells

IL-27 mediates HLA class I up-regulation, which can be inhibited by the IL-6 pathway, in HLA-deficient Small Cell Lung Cancer cells. Supplementary Figures and Table. (DOCX 2856 kb

ABCB1 Structural Models, Molecular Docking, and Synthesis of New Oxadiazolothiazin-3-one Inhibitors

Docking methods are powerful tools for in silico screening and drug lead generation and optimization. Here, we describe the synthesis of new inhibitors of ABCB1 whose design was based on construction and preliminary confirmation of a model for this membrane transporter of the ATP-binding cassette family. We chose the strategy to build our three-dimensional model of the ABCB1 transporter by homology. Atomic coordinates were then assayed for their reliability using the measured activity of some oxadiazolothiazin-3-one compounds. Once established their performance by docking analysis, we synthesized new compounds whose forecasted activity was tested by MTT and cytofluorimetric assays. Our docking model of MDR1, MONBD1, seems to reliably satisfy our need to design and forecast, on the basis of their LTCC blockers ability, the inhibitory activity of new molecules on the ABCB1 transporter

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

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

<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

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

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

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