Main characteristics of study participants.

<p>PSA, prostate-specific antigen; CI, confidence interval; NA, not available.</p><p>Main characteristics of study participants.</p

SEPT9_i1 staining in prostate cancer metastases.

<p>Metastatic prostate cancer lesions from bone marrow, lymph node and bone were immunostained with SEPT9_i1. Left panels are low (x100) magnification (LM) (scale bar 100 μm) and right panel are high (x200) magnification (HM) (scale bar 50 μm). Note high level of SEPT9_i1 staining in all metastases.</p

Correlation between "patients' characteristics" and SEPT9_i1 staining.

<p>PSA, prostate-specific antigen.</p><p>Correlation between "patients' characteristics" and SEPT9_i1 staining.</p

Characterization of SEPT9_i1 antibodies and scoring of SEPT9_i1 staining intensity.

<p>HEK-293T embryonic human kidney cells were transiently transfected with Flag-SEPT9_i1 construct or empty vector (EV). (A) Whole cellular extracts were prepared and analyzed by 4–20% SDS-PAGE and immunoblotting (IB) with antibodies to Flag (1:2000), SEPT9_i1 (1:3000), preimmune serum (1:3000) or SEPT9_i1 antibody (1:3000) pre-incubated with 10 μM of the immunogen peptide for 4 hours. (B) The same cellular extracts were subjected to immunoprecipitation (IP) using anti-Flag antibody and the immuneprecipitates were subjected to 4–15% SDS-PAGE and then immunoblotted with anti-Flag or anti-SEPT9_i1 antibodies. (C) Representative SEPT9_i1 staining in human prostate cancer specimens. Score 0: no SEPT9_i1 staining, 1: low SEPT9_i1 staining 2: medium SEPT9_i1 staining and 3: high SEPT9_i1 staining. Magnification x200, scale bar 50 μm.</p

Gleason score correlation with SEPT9_i1 staining intensity.

<p>Mean SEPT9_i1 staining intensity ± SE of each Gleason score group (score 5: 3 patients, score 6: 8 patients, score 7: 19 patients, score 8: 3 patients, score 9: 6 patients and score 10: 2 patients) was calculated using either visual scoring (A) or automated image analysis with the ARIOL-SL50 system (B).</p

COUP-TF1 expression in the prostate of pre-pubertal mice.

<p>Prostates from pre pubertal Balb/c mice at ages 3, 5, 7 weeks were obtained and an immunohistochemical stain for COUP-TF1 was preformed. For each age group at least 4 mice were analyzed. A. stromal cell staining for COUP-TF1 was quantified by a pathologist as percent of stromal cells stained. An average of all lobes was calculated for each mouse and used for further analysis. P-value was calculated using student's t-test compared to age 3 weeks. B. A normal 3 week old mouse prostate was stained for AR and c. COUP-TF1. Serial sections from the same gland are shown. Epithelial cells are positive for AR and negative for COUP-TF1. Peri-epithelial stromal cells, between blue and yellow lines, are negative for AR and positive for COUP-TF1. Stromal cells more distant from the gland, between yellow and green lines, are positive for AR and negative for COUP-TF1. Similar inversely correlated staining patterns were seen in all slides examined.</p

Over represented Transfac motifs.

<p>Over represented Transfac motifs.</p

Genome-Wide Analysis of Androgen Receptor Targets Reveals COUP-TF1 as a Novel Player in Human Prostate Cancer

<div><p>Androgen activity plays a key role in prostate cancer progression. Androgen receptor (AR) is the main mediator of androgen activity in the prostate, through its ability to act as a transcription mediator. Here we performed a genome-wide analysis of human AR binding to promoters in the presence of an agonist or antagonist in an androgen dependent prostate cancer cell line. Many of the AR bound promoters are bound in all examined conditions while others are bound only in the presence of an agonist or antagonist. Several motifs are enriched in AR bound promoters, including the AR Response Element (ARE) half-site and recognition elements for the transcription factors OCT1 and SOX9. This suggests that these 3 factors could define a module of co-operating transcription factors in the prostate. Interestingly, AR bound promoters are preferentially located in AT rich genomic regions. Analysis of mRNA expression identified chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) as a direct AR target gene that is downregulated upon binding by the agonist liganded AR. COUP-TF1 immunostaining revealed nucleolar localization of COUP-TF1 in epithelium of human androgen dependent prostate cancer, but not in adjacent benign prostate epithelium. Stromal cells both in human and mouse prostate show nuclear COUP-TF1 staining. We further show that there is an inverse correlation between COUP-TF1 expression in prostate stromal cells and the rising levels of androgen with advancing puberty. This study extends the pool of recognized putative AR targets and identifies a negatively regulated target of AR – COUP-TF1 – which could possibly play a role in human prostate cancer.</p> </div

ChIP on chip analysis of AR bound promoters in LAPC4 cells.

<p>LACP4 cells were androgen deprived for 72 hours and then treated with vehicle (ethanol), a synthetic androgen (R1881) or the AR antagonist flutamide. Cells were fixed 16 hours after treatment and ChIP on chip analysis was performed to identify AR bound promoters. A. Number of AR bound promoters in each treatment group, and overlap between groups are presented in the Venn diagram. In each treatment group the sequence of the promoter's probe in the array was analyzed for presence of the classical AR response element (ARE) or the half site. Shown are frequencies of sequences containing the ARE or half site found in each treatment group and the frequency in all of the probes on the array (background). A p-value of enrichment was calculated based on group frequency compared to the background of the array using standard student's t-test. Inset box shows the classical AR response element sequence. B. Table showing the enrichment of ARE and half site in overlaps between groups. For each pair of experimental conditions the frequency of ARE and half site is calculated in overlapping promoters and in all promoters of both groups. P-value of enrichment in overlapping promoters compared to all promoters is calculated using hypergeometric distribution. C. Co-immunoprecipitation of AR and SOX9 in LAPC4 cells.</p

Over represented Gene Ontology Categories.

<p>Over represented Gene Ontology Categories.</p