15 research outputs found

    Gene expression analysis in NB and HeLa cells and correlation analysis.

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    <p>(A) Relative gene expression analysis of the <i>ALK</i>, <i>PHOX2B</i> and <i>PHOX2A</i> genes, carried out in a panel of NB and in HeLa cell lines by real-time RT-qPCR using a pool of normal tissue RNAs as reference sample (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013108#s4" target="_blank">Methods</a>), shows over-expression of the three genes in all but two NB cell lines tested (GI-ME-N and ACN). (B) X-Y Plots showing a significant correlation between the expression level of <i>PHOX2B</i> and <i>PHOX2A vs.ALK</i> (left) and <i>PHOX2Avs. PHOX2B</i> (right) genes in the analyzed cell lines. Pearson's correlation coefficient indicates a very significant correlation of the three transcription levels <i>vs.</i> each other. Values are the mean ± s.d. of N = 3 independent RT-qPCR analyses performed in triplicate.</p

    siRNA-mediated silencing of <i>ALK, PHOX2B</i> and <i>PHOX2A</i> in NB cells.

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    <p>Effects on the transcription level of the <i>ALK</i> (left side graphs), <i>PHOX2B</i> (middle graphs) and <i>PHOX2A</i> (right side graphs) genes after knock-down of the same genes in SHSY-5Y (A), IMR-32 (B) and HTLA-230 (C) cells. Gene-specific knock-down, evaluated 48 hours post-transfection by real-time RT-qPCR analysis, is very effective but also <i>PHOX2</i>-directed siRNAs are able to downregulate <i>ALK</i> at a similar extent (**: <i>P</i><0.01; ***: <i>P</i><0.001). Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate. (D) Gene silencing was confirmed at 72 hours post-transfection by Western blot.</p

    Effects of mutagenesis of ATTA 3 and ATTA 4/5 on the PHOX2B-mediated <i>ALK</i> trans-activation.

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    <p>Left side: schematic representation of the three constructs carrying all the ATTA boxes functional (wt, all four black circles), the ATTA 3 disrupted (ATTA 3 mut, one white circle) or both the ATTA 4 and 5 disrupted (ATTA 4/5 mut, two white circles). Right side: induction of the <i>ALK</i> promoter containing the mutant ATTA 3 and ATTA 4/5 in HeLa cells co-transfected with the PHOX2B expression plasmid are expressed as percentage of the Luciferase activity obtained by cells co-transfected with PHOX2B and the <i>ALK</i> promoter (wt) vectors (wt, arbitrary value = 100). Values are the mean ± s.d. of N = 3 independent experiments (*: <i>P</i><0.05).</p

    PHOX2B effect on the <i>ALK</i> promoter sequentially deleted plasmids.

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    <p>A) Schematic representation of deleted plasmid inserts, progressively shorter from the entire wt <i>ALK</i> promoter region considered (−671 bp), down to the so called deletion 2 (del2; −351 bp), and to the so called deletion 3 (del3; −31 bp) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013108#pone-0013108-g004" target="_blank">figure 4A</a>). The promoter (grey bar), the 5′UTR (black bar) and the ATTA boxes (black circles) are shown. B) Activity of the <i>ALK</i> promoter fragments, expressed as percentage of the activity of the wt construct. Values are the mean ± s.d. of N = 3 independent experiments performed in HeLa cells. C) PHOX2B-mediated induction of the <i>ALK</i> promoter deleted plasmids, expressed as fold increase of the Luciferase activity obtained with respect to the use of the empty vector (pcDNA3.1) on the wt promoter. Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate in HeLa cells.</p

    <i>In vitro</i> interaction of PHOX2B with the <i>ALK</i> promoter.

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    <p>A) EMSAs were performed using probes containing one of the ATTA sites of the region under analysis (ATTA 1, ATTA 2, ATTA 3 and the complex ATTA 4/5). Each labeled probe was incubated in the absence of nuclear extracts (lane 1), with IMR-32 nuclear extracts (lanes 2–4) or the <i>in vitro</i> expressed PHOX2B-Myc fusion protein (lanes 5–7). As negative control the oligonucleotides were also incubated with the <i>in vitro</i> reaction performed using the empty vector pcDNA3.1 M/H (lane 8). The competition experiments were performed in the presence of a molar excess of the unlabeled oligonucleotides (lanes 3 and 6). The anti-PHOX2B or the anti-c-Myc antibodies were added to the samples run in lanes 4 and 7, respectively. On the left, the arrows indicate the specific retarded bands detected; on the right, one or two asterisks indicate the supershifted complexes containing PHOX2B obtained by incubation of IMR-32 nuclear extracts with the anti-PHOX2B antibody (*) or the <i>in vitro</i> expressed protein with the anti-cMyc antibody (**), respectively. The free probes are shown at the bottom of the gels. B) ChIP assay. Chromatin extracted from IMR-32 cells was immunoprecipitated using the antibody against PHOX2B; pre-immune chicken IgY and the anti-acetylated histone H4 antibodies were used as negative and positive controls, respectively. The input represent 0,5% of the total chromatin extract. The precipitated DNA has undergone PCR amplification by using primers bordering the ATTA 3 and the ATTA 4/5 boxes in the <i>ALK</i> promoter.</p

    Forced over-expression of <i>ALK, PHOX2B</i> and <i>PHOX2A</i> in HeLa and NB cell lines.

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    <p>A) Transcription levels of the <i>ALK</i>, <i>PHOX2B</i> and <i>PHOX2A</i> genes were evaluated in HeLa cells 48 hours post-transfection with the corresponding gene-specific cDNA expressing vectors by real-time RT-qPCR. Besides the dramatic increase of gene transcripts by each respective transfectants, <i>ALK</i> expression results enhanced by the <i>PHOX2</i> genes over-expression. Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate. B) Upper (I) and lower (II) lanes from immunofluorescence analysis report examples of HeLa cells transfected with the PHOX2B-Myc expression construct. From left to right images show DAPI stained cell nuclei (blue), staining for the PHOX2B-Myc protein (green), and staining for the ALK protein (red). The most distal image is the merge of the three nearby figures. C) Western blot evaluating protein amounts of ALK and PHOX2B in HeLa cells at 72 hours post-transfection with gene-specific cDNA constructs. A consistent transcript increment of each gene is observed but ALK starts to be expressed also following the forced expression of PHOX2B-Myc protein. Gel was loaded with 100 µg of total protein extracts except for evaluation of ALK in <i>ALK</i>-transfected cells and PHOX2B in <i>PHOX2B</i>-transfected cells, for which 1∶10 (10 µg) of protein extracts was loaded to avoid over-saturation of autoradiograph films. D) Western blot evaluating protein amounts of ALK in ACN cells (left) at 72 hours post-transfection with the PHOX2B-Myc construct, in a clone of IMR-32 cells stably expressing the same PHOX2B-Myc fusion protein (right). A marked increment in ALK expression is detected following PHOX2B-Myc expression in transient –transfected ACN cells compared to both native and mock-transfected cells and in a clone of IMR-32 cells, stably expressing PHOX2B-Myc, compared to native cells. An anti-cMyc antibody was specifically used to distinguish PHOX2B-Myc fusion protein from endogenous PHOX2B of NB cells (lower blots). E) Two stable IMR-32 clones, one negative (104) and one positive (49) for PHOX2B-Myc expression were analyzed for ALK expression (upper panels); expression of the fusion PHOX2B-Myc protein was assessed by Western Blot using anti –cMyc (middle panels) and the anti-actin antibodies (bottom panels).</p

    Effect of ATTA 4/5 disruption in competition of PHOX2B binding.

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    <p>IMR32 nuclear extracts were incubated with the ATTA4/5 probe (lane 2) and competition obtained by adding an excess of: a wild type (wt) probe (lane 3), a probe mutated in both ATTA 4 and ATTA 5 sites (lane 4) or in each of them (lanes 5–6). Incubation without nuclear extracts was regarded as negative control (lane 1).</p

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

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    <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

    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.

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    <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
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