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

<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

Modulation of RET-independent genes.

<p>Histogram bar graph showing the relative transcript levels of genes that, regardless of treatment with GDNF and GFRα1, are differently modulated in PBMCs from healthy donors and HSCR patients. The amounts of mRNA either in freshly purified (left part of the graph) or treated (right part of the graph) of PBMCs of healthy donors (white bars) and HSCR patients (black bars) were detected by Taqman Low Density Array (TLDA) card.</p

Expression of RET receptor on immune cells from HSCR patients associated with pathogenic mutations of RET gene.

<p>Summary graph of statistical histogram bars (upper panel) and dot plots (lower panel) showing MFI values of RET expressed on monocytes, T, B and NK lymphocytes from 46 HSCR patients stratified on the basis of individuals either carrying (gray histogram bars in upper panel and black symbols in the lower panel) or not carrying (empty histogram bars in upper panel and empty symbols in the lower panel) pathogenic RET mutation. 4 HSCR patients were excluded from the analysis because they were carrying mutations of the RET gene with uncertain effects.</p

Levels (pg/ml) and standard deviation (SD) of soluble inflammatory cytokines and chemokines measured in the supernatant of PBMCs either in the absence (italic) or in the presence (bold italic) o treatment with GDNF and GFRα1.

<p>Levels (pg/ml) and standard deviation (SD) of soluble inflammatory cytokines and chemokines measured in the supernatant of PBMCs either in the absence (italic) or in the presence (bold italic) o treatment with GDNF and GFRα1.</p

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

<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

Correlation between the RET receptor expression and RET transcripts on four different cell lines.

<p>(A) Flow cytometric histogram graphs showing the MFI levels of expression of RET receptor (black line) of 4 different cell lines. Gray shaded histograms represent the isotype controls. (B) Histogram bar graph showing the number of RET mRNA copies produced by the same cell lines displayed in panel A and analyzed within the same time-frame in culture. Values are normalized on SK-N-MC cell line of one experiment and are reported as fold increased in expression (2^−ΔCt) as mean of three independent experiments. Of note, the level of RET receptor expressed on cell membrane significantly correlated with the amount of RET transcripts, as assessed by the Spearman rank test for correlation.</p

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

<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.

<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

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

<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.

<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