Effect of selected miRNAs on the expression of endogenous <i>ACVR1/Alk-2</i> mRNA.

<p>Selected miRs were transfected in the indicated cell lines and expression of <i>ACVR1/Alk-2</i> mRNA was measured by RT-qPCR and compared to the expression level of <i>ACVR1/Alk-2</i> transcript from cells transfected with a negative scrambled pre-miR control considered as 100. (<b>A</b>) miR148b; (<b>B</b>) miR365; (<b>C</b>) miR26a. Differences between the effect on <i>ACVR1/Alk-2</i> mRNA of specific miR compared to that of the Cy3-labeled negative control were evaluated by performing a <i>Student’s t-test</i> with <i>p</i><0.05*, <i>p</i><0.01**, <i>p</i><0.001***.</p

Effect of the presence of the <i>ACVR1/Alk-2</i> 3′UTR sequence on reporter gene expression.

<p>A) The whole <i>ACVR1/Alk-2</i> 3′UTR sequence and both the proximal and the distal derived fragments, carrying the miR binding sites containing module (3′UTR M) and the ARE sequences containing segment (3′UTR A), respectively, were subcloned into the pGL3 Promoter vector downstream the Luciferase coding sequence. B) After transient transfection in the indicated cell lines, reporter activity was measured and normalized against the pRL-SV40 expressing the Renilla Luciferase gene for transfection efficiency. Reporter gene activity of the construct carrying the <i>ACVR1-Alk2</i> 3′UTR (dark grey bars), or the M (pGL3+3′UTR M), and the A (pGL3+3′UTR A) fragments are reported as percentage of the activity obtained with the vector lacking the 3′UTR sequence (pGL3-PV, black bars) considered as 100%. Results are from at least two independent transfection experiments made in triplicate (RLU, Relative Luciferase Units). Two-tailed <i>Student</i>’<i>s t-test</i> was performed and significant differences in comparison to the activity of the pGL3-PV empty vector are given as: <i>p</i><0.05*; <i>p</i><0.01**; or <i>p</i><0.001***.</p

Effect of selected miRs on the expression of the <i>ACVR1/Alk-2</i> 3′UTR reporter construct.

<p> Relative Luciferase activity obtained after co-transfection of the <i>ACVR1/Alk-2</i> 3′UTR reporter construct with either a scrambled mir Cy3-conjugated as negative control (Black histograms), or each of the indicated miR (light grey), mir148b (A), mir365 (B) and mir26a (C) or with the corresponding anti-miR (dark grey histograms). The reporter gene activity measured in presence of the Cy3-miR negative control is considered as 100 for each independent experiment, RLU, Relative Luciferase Units. The statistical significance of the effect of miR compared to that obtained with the non targeting negative control was evaluated by <i>Student’s T-test</i> analysis, with <i>p</i><0.05*; <i>p</i><0.01**; or <i>p</i><0.001***.</p

Predicted miR recognition sequences in the 3′UTR of the <i>ACVR1/Alk-2</i><b> gene.</b>

<p>For each of the selected miRs, duplexes between miRNA sequences (middle) and wild-type (bottom) 3′UTR or harboring the corresponding mutated seed (top) are shown. Numbers on the left side indicate the matching starting position in the <i>ACVR1/Alk-2</i> 3′UTR, G:U wobble (<b>:</b>) and Watson-Crick pairing (<b>|</b>) are also indicated. miRVS scores rank the efficiency of the predicetd binding sites, good scores are ≤-0.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050958#pone.0050958-Vasudevan1" target="_blank">[33]</a>.</p

<i>ACVR1/Alk-2</i> mRNA is well conserved among species and is unstable in presence of ActD.

<p>A) Output of the comparative genomic analysis obtained with a dedicated software available at the Vista Genome Browser (<a href="http://genome.lbl.gov/vista/index.shtml" target="_blank">http://genome.lbl.gov/vista/index.shtml</a>, tools for comparative analysis*). Non coding Conserved sequences corresponding to the <i>ACVR1/Alk-2</i> 3′UTR are shown as light blue peaks, and are shown relative to their position on human chr2 (March 2006, release) compared to the mouse (first track) rat (second track) and chicken (third track) genomes, respectively, the percentage of identity is indicated on the vertical axis. (*Frazer KA, Pachter L, Poliakov A, Rubin EM, Dubchak I. VISTA: computational tools for comparative genomics. Nucleic Acids Res. 2004 Jul 1;32 (Web Server issue):W273-9). B) and C) C2C12 cells were treated with the ActD transcription inhibitor with the doses and for the time points indicated. <i>ACVR1/Alk-2</i> mRNA was measured by semi-quantitative (B) and quantitative RT-PCR (RT-qPCR, <b>C</b>). Two-tailed student′s t-test was performed. Significant differences in comparison to untreated cells are given as: <i>p</i><0.05*; <i>p</i><0.01**; or <i>p</i><0.001***.</p

Binding site specific mutagenesis prevents the modulation of <i>ACVR1/Alk-2</i> expression operated by the selected miRs.

<p> Mutagenesis of miRs binding sites was introduced in the two seed sequences predicted for mir148b (A) and mir365 (B), indicated as Seed1 and Seed 2, and in the mir26a seed sequence (C). Site specific mutagenesis was also performed to abolish the first AUUUA element contained in the mir26a pairing region (ATTTA mut), alone or in combination with the mutation of the mir26a seed sequence (double mutant mir26 mut/ATTTA mut). Each pair of bars represents Luciferase activity in the presence of negative control miR (-), considered as 100%, or the specific miR (+). <i>Student’s t-test</i> was applied to evaluate differences between the effect of specific mir compared to that of the negative control for each construct, <i>p</i><0.05*, <i>p</i><0.01**, <i>p</i><0.001***.</p

Expression of RET receptor on immune cells from HSCR patients associated with a single nucleotide polymorphism at exon 2.

<p>Summary graph of statistical dot plots showing MFI values of RET receptor expressed on lymphocytes and monocytes from 50 HSCR patients with medians (horizontal black bars) either in the absence (A) or in the presence (B) of an association analysis stratification based on the genotype at the exon 2 of RET gene (SNP rs1800858). We did not detect any statistically significant association between the phenotypic distribution of RET receptor on immune cells with RET genotype at exon 2 SNP.</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

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