Western blotting analysis of proteasomal degradation and stability of Ile60Asn parafibromin.

<p><b>A</b>. Western blotting for parafibromin (with anti-<i>CDC73</i> antibody) in HeLa cells in absence (−) or in presence (+) of the proteasome inhibitor MG132. <b>B</b>. Western blotting for parafibromin in HeLa cells after treatment with CHX (0 = no treatment) and harvesting at the indicated time points. <b>C</b>. Graphical representation of the stability of wild-type parafibromin (WT-CDC73) and Ile60Asn mutant at different time points after treatment with CHX.</p

Western blotting for parafibromin and tGFP 48 hours post-transfection.

<p>Immunoblotting for parafibromin in HEK293A (<b>A</b>) and HeLa cells (<b>B</b>) transfected with Ile60Asn-tGFP-fused parafibromin <b>(Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or empty vector (<b>tGFP</b>). Nontransfected cells (<b>Neg ctrl</b>) have been used as a negative control. <b>C</b>. Western blotting with an anti-tGFP antibody has been performed as a control on Ile60Asn and wild-type-parafibromin-transfected cells and on negative control cells. ∼61 kDa: band showing proteolytic degradation of the exogenous parafibromin-tGFP, which generated a protein fragment of approximately the same size of endogenous parafibromin. ∼26 kDa: band corresponding to unconjugated tGFP molecular weight.</p

<i>CDC73</i> sequencing and anti-parafibromin immunohistochemistry in the mandibular tumor.

<p><b>A.</b> Electropherograms of <i>CDC73</i> exon 2 PCR amplicons from tumor DNA after subcloning. Upper panel: allele carrying only the somatic c.179 T>A transversion; lower panel: allele carrying the germline c.(136_144)del5 frameshift mutation. The nucleotides in the wild- type sequences involved in the frameshift deletion and in the single nucleotide substitution are underlined with black lines. <b>B</b>, <b>C</b>. Anti-parafibromin immunostaining in the ossifying fibroma of the jaw. Original magnifications: 10X (A) and 63X (B). <b>D</b>, <b>E</b>. Anti-parafibromin immunostaining in positive controls: human normal parathyroid tissue (<b>D</b>) and sporadic parathyroid adenoma with wild-type <i>CDC73</i> (<b>E</b>). Original magnification: 10X.</p

Real-time RT-PCR for <i>CDC73</i> mRNA in transfected and nontransfected HEK293A cells.

<p>Histogram representing the <i>CDC73</i> mRNA fold change in HEK293A cells transfected with Ile60Asn-tGFP-fused parafibromin (<b>Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or empty vector (<b>tGFP</b>). Nontransfected cells (<b>Neg ctrl</b>) have been used as a negative control.</p

Western blotting for cyclin D1 and c-myc 48 hours post-transfection in HEK293A cells.

<p>Immunoblotting for cyclin D1 and c-myc in HEK293A cells transfected with Ile60Asn-tGFP-fused parafibromin (<b>Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or tGFP-vector (<b>tGFP</b>). Nontransfected HEK293A cells (<b>Neg ctrl</b>) have been used as a negative control.</p

Cell proliferation assay.

<p>Cell counts at three different time points (24, 48 and 72 hours) after transfection of HeLa cells with Ile60Asn-tGFP-fused parafibromin (<b>Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or tGFP-vector (<b>tGFP</b>).</p

Crude extract of <i>Origanum vulgare</i> L. induced cell death and suppressed MAPK and PI3/Akt signaling pathways in SW13 and H295R cell lines

<p>Oregano (<i>Origanum vulgare</i> L.) is a common aromatic plant used in Mediterranean and Asian Regions for treating respiratory diseases, painful menstruation, rheumatoid arthritis, etc. Recently its role as an anticancer plant has been suggested, although oregano has been never evaluated into adrenocortical tumour cell models. This study analysed for the first time the anticancer effects of a crude extract of wild mountain oregano (<i>Origanum vulgare</i> L.) in SW13 and H295R cell lines. The crude extract was characterised by GC/MS and the toxic effects of oregano were first analysed by brine shrimp lethality assay. Our findings demonstrated that oregano decreased cell viability, survival, modified cell cycle and induced cell death (through necrotic process) and that the effects can be attributed to a blockade of MAPK and PI3 K/Akt pathways. These results suggest that oregano extract exerts anticancer activities in adrenocortical tumour cell lines, providing evidence for further research in higher models.</p

<i>LAT1</i>, <i>LAT2</i> and <i>GLUT1</i> genes expression in PHEO.

<p>(<b>A</b>) Box plots of relative qPCR gene expression measurements of <i>LAT1</i>, <i>LAT2</i> and <i>GLUT1</i> in PHEO specimens and the relative paired normal tissues. Each value was referred to a pool of normal thyroid tissues that was set to 1. Each dot represents a single sample. (<b>B</b>) and (<b>C</b>) Associations between the <i>LAT1</i> and <i>GLUT1</i> (<b>B</b>) and the <i>LAT2</i> and <i>GLUT1</i> (C) gene expressions in PHEO specimens, by Spearman’s rank correlation. (<b>D</b>) and (<b>E</b>) Box plots representing the correlation between the <i>LAT1</i> expression levels in PHEO, dichotomized according to the median value, with the urinary levels of adrenaline (<b>D</b>) and noradrenaline (<b>E</b>). (<b>F</b>) Box plots representing the correlation between LAT2 expression levels in PHEO, dichotomized according to the median value, with the urinary levels of adrenaline (left) and noradrenaline (right). Boxes indicate the range from lower to upper quartile values, with the line inside the box representing the median. The vertical lines mark the highest and lowest value observed within a distance of 1.5 times the inter-quartile range from the bottom and the top of the boxes, respectively.</p

LAT1 immunohistochemistry.

<p>(<b>A</b>-<b>B</b>) Pheochromocytoma, strong cytoplasmic staining of LAT1 with membranous enhancement sparing of the neoplastic nuclei and normal adrenal tissue <b>(C)</b>. (<b>D-E</b>) Medullary thyroid carcinoma, strong cytoplasmic staining of LAT1 with membranous enhancement sparing of the neoplastic nuclei and normal thyroid tissue <b>(F)</b>. (<b>A-C and F</b>) 20× original magnification; (<b>D-E</b>) 10× original magnification.</p

Clinical features of patients with pheochromocytoma.

<p>Clinical features of patients with pheochromocytoma.</p