LAT1, LAT2 and GLUT1 genes expression in MTC.

<p>(A) Box plots of relative qPCR gene expression measurements of <i>LAT1</i>, <i>LAT2</i> and <i>GLUT1</i> in MTC 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> (<b>C</b>) gene expressions in MTC specimens, by Spearman’s rank correlation. (<b>D</b>) Frequency chart representing the Fisher’s exact test correlation between the LAT1 expression levels in MTC, dichotomized according to the median value, with the tumor size dichotomized for the size of 10 millimeters.</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

Western blot analysis and corresponding <i>LAT1</i> mRNA expression level.

<p>Representative Western blot analysis of normal (N)/tumoral match-pair samples for the expression of LAT1 in PHEO (<b>A</b>) and MTC (<b>B</b>) samples. Samples were corrected for protein loading by β-actin. Corresponding bar graphs for relative mRNA expression level of <i>LAT1</i> in (C) PHEO and MTC (D).</p

Clinical features of patients with medullary thyroid cancer.

<p>Clinical features of patients with medullary thyroid cancer.</p

Clinical features of patients with pheochromocytoma.

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

Constructs used in transfection experiments.

<p>(A) Either the wild type, the c.-456_-453delCCTT or the c.-469C>T containing 5′UTRs were cloned upstream the firefly luciferase gene. (B) The wild type/c.-456_-453delCCTT 5′UTR were subcloned upstream the <i>CDKN1B</i> gene. (C) The introduction of the c.-428A>T substitution in the two former plasmids reported in panel A restores uORF length and intercistonic distance. (D) Constructs reported in panel B were mutated introducing a c.-74insC leading to the translation of a chimeric protein in the double mutant.</p

The c.-456_-453delCCTT affects protein translation by reducing reinitiation.

<p>Schematic representation of the <i>CDKN1B</i> 5′UTR in the wild type (A) and in the c.-456_-453delCCTT carrier (B). The uORF coding sequence is shown as grey box. Electropherograms for germline (G) and tumor (T) DNA are shown.</p

Effect of the c.-428A>T restoring mutation.

<p>In both GH3 and HeLa cell lines c.-428A>T almost restores the modulation properties of the <i>CDKN1B</i> wild type uORF (A). Panel B shows the primary sequence differences in the wild type uORF compared with the -456_-453delCCTT+c.-428A>T.</p

Polysome profiling of lymphoblastoid cells from the -456_-453delCCTT mutation carrier.

<p>(A) Representative absorbance profile for RNA separated by velocity sedimentation through a 15–50% sucrose gradient. The positions of the 40S, 60S, 80S, and polysomal peaks are indicated. (B) The levels of <i>CDKN1B</i> mRNA (wt and c.-456_-453del) in each gradient fraction were measured by quantitative real-time PCR and plotted as a percentage of the total <i>CDKN1B</i> mRNA levels (wt or c.-456_-453del, respectively) in that sample. Data represent three independent experiments, with mean ± SD reported. (C) The levels of both alleles in each fraction were expressed as relative quantities calculated from differences in Cq values between mRNA and genomic DNA for removing the intrinsic variation between the two qPCR assays. Data represent the mean of three independent experiments ± SD.</p