Most common recurrent copy number alterations detected by a-CGH in CDC73-mutated parathyroid tumors (T2–T8) with corresponding region of LOH.

*<p>Genes written in bold are known cancer genes according to Cancer Gene Census, Welcome Trust, Sanger Institute.</p

Figure 2

<p>(A) Bar graph showing predicted DNA copy number of the <i>CDC73</i> gene using TaqMan DNA copy number analysis. Each bar represents one parathyroid sample and the height of the bar represents the predicted <i>CDC73</i> gene DNA copy number. T2, T3 and T4 had 1 copy of <i>CDC73</i> gene, while T6 and T8 had more than 2 copies. N1, N2 and N3 refer to the normal parathyroid samples used as calibrators. (B) Individual value plot illustrating the methylation density at the three analyzed CpG dinucleotides of the <i>HPRT2</i> promoter. Each red dot represents one CpG site in one parathyroid tumor numbered from T2–T8 along with the three normal references denoted as N1, N2 and N3.</p

LOH karyogram.

<p>Karyogram showing all LOH events detected in (A) the parathyroid carcinomas (T6–T8) and in (B) the parathyroid adenomas (T2–T5) using a 250K SNP array.</p

Sequencing chromatograms of <i>CDC73</i> mutations in parathyroid tumors: T1 (IVS1+1 G>C), T5 constitutional (c128-IVS1+1 delG) and T5a (c.71del 72–90).

<p>Wild-type (WT) sequences are indicated above the mutated sequences.</p

Characteristics of the 9 parathyroid tumors.

<p>M = Male, F = Female, Ex = Exon, Int = Intron, n.a = not available.</p><p>LOH =  Loss of heterozygocity, n.d. = not determined.</p>*<p>CDC73/HRPT2 mutations were previously published for cases T2–T4, and T6–T8 (References <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046325#pone.0046325-Carpten1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046325#pone.0046325-Shattuck1" target="_blank">[5]</a>).</p><p>The constitutional mutation in T5 was revised from a previous publication (Reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046325#pone.0046325-Juhlin4" target="_blank">[30]</a>).</p

HeatMap representing unsupervised hierarchical whole genome clustering incorporating all the CNAs detected by a-CGH (with the exclusion of T1 and T5a).

<p>Three clusters were identified. One cluster grouped the carcinomas T6, T7, and T8, another cluster included T2, T3 and T5b, while T4 grouped separately. Grey refers to no changes, red to gain and blue to loss.</p

Localisation of PRLr expression to lysosomes in normal parathyroid rim and to enlarged lysosomes in parathyroid tumour tissue.

<p>A) Immunohistochemistry with PRLrI showing “ring-like” cytoplasmic structures and cytoplasmic reactivity. B) Immunohistochemistry of PRLrI showing cytoplasmic granuale and cytoplasmic reactivty. C and D) Analysis of “ring like” structures and cytoplasmic granulae by flourescent immunohistochemistry. Images show one parathyroid tumour (C) and normal parathyroid rim (D), stained with DAPI (blue), anti-PRLrI (red, upper right) and anti-SCARB2 (green lysosomal marker, lower left) separately and in overlay (lower right and upper left).</p

Immunohistochemical analysis of PRLr expression using the PRLrI antibody

<p>. The photomicrographs show parathyroid tumour tissues (A–C) and negative control (D). Parathyroid tumours are shown with immunostaining of cytoplasmic granulae and cytoplasm (A), of cytoplasm only (B), and of cell membrane and cytoplasm (C).</p

qRT-PCR analysis for <i>PRLR</i> expression in normal parathyroid tissue and parathyroid tumours.

<p>Box-plots show<i>PRLR</i>-total mRNA expression after normalization in normal parathyroid tissue and parathyroid tumours (A), and normal parathyroid and other normal tissues (B). The arbitrary value of 1.0 indicating the expression level in MCF-7 cells is indicated.</p

Analysis of PTH secretion and intracellular Ca²⁺ after prolactin treatment.

<p>A) Measurements of PTH secretion from parathyroid adenoma cells upon treatments with prolactin at 100 µg/L or 200 µg/L. Results from four independent experiments are shown. B) Example of a measurement of intracellular Ca²⁺ in parathyroid adenoma cells treated with 100 µg/L followed by 200 µg/L prolactin, as indicated by vertical red lines.</p