Knock down of NDRG1 expression by shRNAs.

<p>Western blots detect reduced NDRG1 expression in the breast cell lines ME16C2 and SUM102 expressing the shRNAs NDRG1si4 (si4) and NDRG1si7 (si7), transduced in parallel experiments (A and B) compared to control cells (C) transduced with the empty vector pSiRPG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087268#pone.0087268-Storvold1" target="_blank">[27]</a>. α-tubulin was used as loading control.</p

Increased NDRG1 expression due to hypoxia or ectopic expression.

<p>(<b>A</b>) Increased NDRG1 expression detected by Western blotting in the breast cell lines MCF-7 and ZR-75-1 grown at 1% O₂ for 24, 48 and 72 hours as indicated, compared to control cells grown at 20% O₂. (<b>B</b>) Western blots document increased NDRG1 expression in cells grown at 20% O₂. The ZR-75-1 cell populations transduced with the NDRG1-cDNA (six biological parallels indicated A–F), are compared to ZR-75-1 transduced with the vector pSiRPG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087268#pone.0087268-Storvold1" target="_blank">[27]</a> without insert (C2 and C3). α-tubulin was used as loading control (<b>A</b> and <b>B</b>). Note that a reduced level of total protein is loaded in the lane containing extract from MCF-7 grown at 1% O₂ for 24 hours. (<b>C</b>) Treeview presentation of the expression of 18 genes in a hypoxia signature gene list <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087268#pone.0087268-Chi1" target="_blank">[24]</a> in MCF-7 and ZR-75-1 cells grown at 1% O₂ for 24 or 48 hours as indicated. (<b>D</b>) Treeview presentation of the hypoxia signature genes in six ZR-75-1 cell populations ectopically over-expressing NDRG1 (indicated A–F). The red and green colours show increased and reduced expression levels, respectively (<b>C</b> and <b>D</b>). The colour intensity is from −3 to 3 indicating the magnitude of the fold change in gene expression between the cell population and its control.</p

Immunofluorescence staining of SUM102 cell cultures.

<p>Colors were applied artificially for graphical purposes. (<b>A</b>) NDRG1 (green) and calnexin (red) in untreated, hypoxia and doxorubicin treated cells. Bar: 5 µm. (<b>B</b>) NDRG1 (green) and EEA1 (red) in hypoxia treated cells. Bar: 10 µm.</p

Tumor_LogR.GCadjusted_qnorm_117

Adjusted Log R Ratio values for the 117 tumour samples studied, after the application of the GC correction method and quantile normalization

Tumor_BAF_117orig

Original B allele frequency values for the 117 tumour samples studied

Diagnoses and histopathologic scores_ASCAT tumours

Histopathomorphological diagnoses and histopathologic score values for all tumours that achieced an ASCAT output (113 of 117 tumours). These diagnoses/scores were used in further analysis of the ASCAT output

Germline_BAF_117

B allele frequency values for the blood samples for each of the 71 dogs in the study

Tumor_LogR_117orig

Original Log R Ratio values for the 117 tumour samples studied

Proportion of probes with LOH in subgroups of CMTs, according to ASCAT analysis.

<p>LOH: Loss of heterozygosity. P: P-value from Kruskal-Wallis test.</p

ASCAT estimate of ploidy in different subgroups of tumours.

<p>Hyperplasias (n = 12), benign (n = 55) and malignant tumours (n = 46). For the graphical presentation, the ploidy estimates were rounded to the nearest whole number.</p