Association between <i>AMY1</i> copy number with BMI z-score in boys and girls.

<p>Linear regression analysis shows a significant reduction of BMI z-score by increasing <i>AMY1</i> relative copy number in boys, but not in girls.</p

General characteristics of the study population.

<p>General characteristics of the study population.</p

Distribution of <i>AMY1</i> copy number in the study population.

<p>Numbers on the top of columns indicate the individuals included in each copy number category. For this figure estimates of copy number have been rounded to the nearest integer.</p

ASE values observed.

<p>ASE values between the 2 dashed lines correspond to less than twofold imbalances in allelic ratios (see Methods). </p

Results of integrative germline analyses performed in this study.

<p>VUS included in-frame deletions, sequence variants with uncertain effect on splicing and missense variants. Evidence of a pathogenic, possibly pathogenic defect in 28 probands with VUS derived from previous studies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081194#pone.0081194.s007" target="_blank">Table S5</a>). In one additional case (360#2916) availability of RNA allowed ASE analysis that helped to characterize a VUS shared also by another proband (986#3487, see Results). ASE analysis was conducted in 22 individuals, including 12 with ascertained germline defect that are not shown in the figure. <i>APC</i> and MUTYH screening was conducted in a subset of patients negative for MMR defects (see Methods). Inclusion of ASE analysis and screening for <i>APC</i> and MUTYH sequence variants in the integrative analyses increased the number of probands with germline alterations detected.</p

Apoptosis in PTJ64i and PTJ86i cells treated with 24 μM GW6471 for 24, 48 or 72 hours.

<p>Values represented in the histograms (<i>top</i>) are the means ±SD of at least two independent determinations (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001). Dot plots (<i>bottom</i>) show representative experiments after a 72-hour treatment with 24 μM GW6471.</p

Immunohistochemical analysis of PPARα in HNPGL tissues.

<p>Tumor sections derived from two different HNPGL patients show positive immunostaining for PPARα in paraganglioma cell clusters, while the supporting stroma is negative. Bar = 80 μm.</p

Growth curves of PTJ64i and PTJ86i cells treated with GW6471.

<p>Cell number was measured over a 72-hour time course treatment with 24 μM GW6471 or with vehicle control. Data shown are the means ±SD of three to five determinations (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001).</p

Effect of GW6471 on PI3K/GSK-3β/β-catenin signaling pathway in PTJ64i and PTJ86i cell lines.

<p>Histograms of normalized densitometric analyses and representative western blotting for PI3K, GSK3β, p-GSK3β and β-catenin in HNPGL cells treated with 24 μM GW6471 for 72 hours are shown. The relative densities of the immunoreactive bands were determined and normalized with respect to GAPDH (loading control) using a semiquantitative densitometric analysis (Kodak ID Image Analysis Software, Rochester, NY, USA). Values are expressed as relative units (RU). Each bar represents the mean ± SD of two up to five independent determinations (*<i>p</i><0.05).</p

Immunofluorescence analysis of PPARs in HNPGL cells.

<p>PTJ64i (<i>left panels</i>) and PTJ86i (<i>right panels</i>) show high levels of PPARα nuclear expression. The fluorescence intensity for PPARα is stronger as compared to that for PPARβ/δ or PPARγ. Bar = 20 μm.</p