Immunocytochemical staining.

<p>A: Native CT1258 cells, B: CT1258-EGFP cells, C: CT1258-EGFP-HMGA2 cells. Approximately 50% of the native CT1258 cell line and of CT1258-EGFP cells showed a HMGA2-positive nuclear labelling. In approximately 70–80% of CT1258-EGFP-HMGA2 cells, a strong and exclusively nuclear labelling for HMGA2 was detectable.</p

Cytogenetic analyses.

<p>Metaphase spreads derived from CT1258 (A), CT1258-EGFP (B) and CT1258-EGFP-HMGA2 (C) cells after GTG-banding. The arrows indicate the centromeric fusions between the canine chromosomes 1 and 5 (der(1;5)) and a large bi-armed marker (mar) consisting of material from chromosomes 1 and 2 being characteristic for the CT1258 cell line.</p

<i>Let-7a</i> real-time PCR analyses.

<p>Relative <i>let-7a</i>/RNU6B expression in native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. Error bars are standard deviations. No statistical significant expression deregulation of <i>let-7a</i> in CT1258-EGFP and CT1258-HMGA2-EGFP was detected when compared to native CT1258 cells. Statistical significant p value was defined as ≤0.05.</p

<i>HMGA2</i> real-time PCR analyses.

<p>Relative <i>HMGA2/HPRT1</i> and <i>HMGA2/GUSB</i> expression in native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. Error bars are standard deviations. *p≤0.05 indicates a statistical significant expression deregulation of <i>HMGA2</i> in CT1258-HMGA2-EGFP cells when compared to native CT1258.</p

BrdU cell proliferation assay.

<p>Measured proliferation of native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. A statistical significant increased proliferation was detected for CT1258 cells expressing the EGFP-HMGA2 fusion protein in comparison to native CT1258 and EGFP expressing CT1258 cells. Each bar represents a mean ± SD, *p≤0.05, ***p≤0.001.</p

<i>HMGA1</i> real-time PCR analyses.

<p>Relative <i>HMGA1/HPRT1</i> and <i>HMGA1/GUSB</i> expression in native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. Error bars are standard deviations. *p≤0.05 indicates a statistical significant increased expression of <i>HMGA1</i> in CT1258-HMGA2-EGFP cells when compared to native CT1258 and CT1258-EGFP.</p

Details of CT1258 chromosomal aberrations.

<p>Detailed presentation of chromosomal aberrations found in metaphases of CT1258, CT1258-EGFP and CT1258-EGFP-HMGA2. Two derivative chromosomes (der(1;5)) and the large bi-armed marker chromosome (mar) were found in each cell line.</p

Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples

<div><p>Immunohistochemistry (IHC) is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1), progesterone receptor (PGR), prolactin receptor (PRLR) and growth hormone receptor (GHR) gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE) was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method.</p></div

List of genes and their respective Accession Number and Probe Set Region used in the QuantiGene 2.0 Plex Assay.

<p>List of genes and their respective Accession Number and Probe Set Region used in the QuantiGene 2.0 Plex Assay.</p

Median of normalized gene expression of ESR1 and PGR in intact (fresh frozen n = 57; FFPE n = 83) and neutered (fresh frozen n = 11; FFPE n = 8) females with malignant mammary tumors.

<p>Median of normalized gene expression of ESR1 and PGR in intact (fresh frozen n = 57; FFPE n = 83) and neutered (fresh frozen n = 11; FFPE n = 8) females with malignant mammary tumors.</p