Additional file 2: Figure S1. of Iroquois homeobox 2 suppresses cellular motility and chemokine expression in breast cancer cells

Gene reporter assay with reporter plasmid containing proximal CCL5 promoter fragment. The reporter plasmid was transfected into BT-549 cells that over express IRX2 and into the control cell line.  For normalization, a co-transfection with the pGL4.74 plasmid containing the Renilla luciferase was performed. Each experiment was performed in triplicate. Error bars represent the standard deviation of the mean. (PDF 18 kb

Elevated Gremlin-1 protein expression in LDS-OECs.

<p>The Gremlin-1 protein expression in LDS-OECs was compared to OECs isolated from sex- and age-matched healthy donors. Immunoblotting followed by quantification revealed that the Gremlin-1 protein amount was increased in all three LDS-OEC clones compared to their respective control.</p

Members of the TGF-β superfamily with altered mRNA expression levels in LDS-OECs compared to healthy controls.

<p>* signal log ratio of LDS-OEC compared to healthy control, determined in microarray analysis; ^† expression fold change of LDS-OEC compared to healthy control, converted from microarray data; ^‡ relative gene expression in LDS-OEC compared to healthy control, determined in quantitative PCR analysis and normalized to <i>GAPDH</i> expression.</p

LDS patients analysed for OEC generation.

<p>* number of generated OEC clones.</p

Gremlin-1 expression on aortic tissue of LDS patients.

<p>Paraffin-embedded aortic tissue specimen of LDS patients (n = 3) were double stained with anti-Gremlin-1 (brown staining) and anti-CD34 (red staining; C) or anti-smooth muscle actin (red staining; B, D–F). Endothelial cells throughout the vessel wall showed expression for Gremlin-1 including ECs of the intima (B) and of vessels within the media (C) and the adventitia (D and in more detail in E). Gremlin-1 positive staining was also observed on smooth muscle cells of the media (B, C) as well as on vessel surrounding smooth muscle cells in the adventitial layer (D, E). In aortic tissue specimen of healthy controls (n = 3), a similar staining pattern without gross differences of staining intensity was observed as shown for a small vessel within the adventitia (F). In A, an isotype control instead of primary antibody was used revealing the specificity of the staining (EC = endothelial cell, SMC = smooth muscle cell; magnification A–D, F: 400×; E: 1000×; scale bar represents 20 µm in A–D and F and 8 µm in E).</p

Patient characteristics of LDS1, LDS5 and LDS11.

<p>* Δ95th identifies the difference of diameters obtained in study patients at baseline minus diameter (cm) at 95th percentile as assessed according to Biaggi <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104742#pone.0104742-Biaggi1" target="_blank">[35]</a>.</p

Heterozygous <i>TGFBR2</i> and <i>TGFBR1</i> mutations identified in LDS patients.

<p>*representing two unrelated families.</p