Experimental design and FoxE1 protein levels after 48 hours of silencing.

<p><i>Panel A:</i> Experimental design of expression array experiments. Two comparisons were done: FoxE1-silenced PCCl3 cells (siFoxE1 PCCl3) <i>vs.</i> scrambled siRNA-treated PCCl3 cells (siScramble PCCl3), and FoxE1-silenced PCCl3 cells <i>vs.</i> wild type PCCL3 cells (wt PCCl3). Each comparison was performed in quadruplicate and using dye swaps. <i>Panel B:</i> Western Blot of extracts from control, siScramble and siFoxE1-treated cells from quadruplicate samples used for microarray analysis. Hybridizations were done with anti-FoxE1 antibodies; anti-tubulin antibodies were used as loading controls.</p

FoxE1 and NF1/CTF binding to and transcriptional activation of the NUE.

<p>A ReChIP assay was used to analyse simultaneous binding of FoxE1 and NF1/CTF proteins to the NUE (panel A). qPCR was done to analyse chromatin immunoprecipitates of PCCl3 cells using FoxE1 and NF1 antibodies. The enrichment of target sequences was calculated as the IP ratio (arbitrary units) relative to the negative control Afm, and normalized to their relative amplification in the input sample. A sequence from the Tg promoter was used as positive control. HeLa cells were transfected with 1 µg of a FoxE1 or NF1/CTF expression vector or the empty vector, and 1.5 µg and 0.1 µg of pNIS 2.8-Luc and CMV-<i>Renilla</i> constructs respectively (panel B). Forty-eight hours after transfection, cells were collected for the measurement of luciferase and <i>renilla</i> levels. Results are shown as the mean±SD of the luciferase levels relative to the non-regulated <i>renilla</i> levels of six independent experiments. (*): p<0.05; (**): p<0.01; (***): p<0.001; two tailed t-test.</p

Summary of FoxE1 microarray results in PCCl3 cells.

<p>Number of statistically significant probes and genes (p<0.005) after Foxe1 silencing; results are shown for each type of comparison performed, as well as for the combined analysis.</p

ChIP experiments for FoxE1 binding to selected genes.

<p>qPCR analysis of chromatin immunoprecipitation performed on PCCl3 cells with FoxE1 antibody. The enrichment of target sequences was calculated as the IP ratio (arbitrary units) relative to the negative control Afm, and normalized to their relative amplification in the input sample. Sequences from the Tg and Tpo promoters were used as positive controls. Two regulatory regions were analysed in the <i>Duox2</i> promoter (called Duox2-1 and Duox2-2). Results are mean ± SEM of two independent experiments, each performed in triplicate.</p

Description of statistically genes significantly (p<0.005) regulated by FoxE1.

<p>Description of statistically genes significantly (p<0.005) regulated by FoxE1.</p

Experimental validation of microarray results by qRT-PCR and western blotting.

<p>Relative expression assessed by means of qRT-PCR of 6 thyroid-specific genes (panel A) and 12 additional genes differentially down- and upregulated in FoxE1-silenced PCCl3 cells (panel B). As FoxE1-dependent positive controls, we evaluated Tpo and Tg mRNA expression levels. Relative gene expression in siFoxE1 samples was calculated using the corresponding siScrambl samples as a reference ( = 1). Results are mean ± SEM of four independent experiments. Total protein extracts were prepared and submitted to western blot analysis to assess the protein levels of FoxE1 and Nis (panel C), and of Cdh1 and Duox2 (panel D). Actin was used as loading control. Representative western blot assays of four independent experiments are shown.</p

Putative FoxE1-NF1/CTF binding sites in the <i>Duox2</i> gene and in the NUE.

<p>Chromosomal location of putative FoxE1 and NF1/CTF binding motifs in the Nis upstream enhancer (panel A) and the <i>Duox2</i> gene (panel B). Oligos used and exons are represented in italics.</p

Gene-gene interaction results for susceptibility to classic Papillary Thyroid Carcinoma.

<p>SH- SNPHarvester; MSH- MegaSNPHunter; Light grey shading indicates interactions selected by each of the algorithms; dark grey green shading indicates the interaction fulfilling the established criteria to pass to stage 2 (replication) (SNP pair selected by at least three methods);</p>*<p>associated P-value; NA- not available (all genotypes combinations classified as neutral).</p

Association with risk of follicular cell-derived thyroid cancer for 9 candidate variants in the discovery and replication stages.

<p>Abbreviations: MAF = minor allele frequency; OR = odds ratio; CI = confidence interval; ESE = Exonic Splicing Enhancers; PTC = Papillary Thyroid Carcinoma; cPTC = classic PTC; fvPTC = follicular variant of PTC; FTC = Follicular Thyroid Carcinoma. The table is sorted by disease subtype and, within each group, by <i>P</i>-value.</p>a<p>Major/minor allele (in controls);</p>b<p>OR and CI were obtained using homozygotes for the most frequent allele in controls as the reference group;</p>c<p><i>P</i>-values are derived from Wald statistics;</p>d<p>Results adjusted for age and gender;</p>e<p>Results adjusted for age, gender and country.</p

Relative expression levels of STK17B in Pax8-silenced cells.

<p>PCCl3 cells were transiently (A) or stably (B) silenced for the transcription factor Pax8 (siPax8). As a control, wild type or siScramble transfected cells were used. The expression levels were assessed by means of qRT-PCR (A, upper panel) or western blot (A, lower panel, and B).</p