Additional file 2: Table S2. of Diagnostic and prognostic value of SHOX2 and SEPT9 DNA methylation and cytology in benign, paramalignant, and malignant ascites

Specification of 25 cancer patients suffering from more than one primary tumor. Other existing primary tumors are listed for patients suffering from more than one primary tumor. (DOC 76 kb

<i>MB</i> exon 5u promoter regulatory networks in tumor cells under different conditions.

<p><b>(a)</b> Hypoxia (1% O₂)-driven upregulation of the promoter in MCF7 cells. HIF1α/β binding to the candidate HRE in region A1 is indicated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.g006" target="_blank">Fig 6a</a>). Regions G and F interact with RNA-PolII. Open chromatin regions are drawn in black (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.g005" target="_blank">Fig 5a</a>). <b>(b)</b> Estrogen (E2)-mediated downregulation of the promoter in MCF7 cells. ERα binding to region A1 may occur at the ERα-half sites and the RUNX1 site (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.g006" target="_blank">Fig 6a</a> for details). The complex bound to region A1 further interacts with p300 and RNA-PolII. Regions C and G also interact with the ERα protein complex, region F associates with RNA-PolII (Figure a in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.s004" target="_blank">S4 Fig</a>). This interaction network recruits a repressor complex to downregulate the transcription of 5u <i>MB</i> transcripts under E2 treatment. Open chromatin regions are marked in black. <b>(c)</b> Androgen (DHT)-driven downregulation of the promoter in LNCaP cells. AR binding to a hypothetical ARE motif in region D1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.g006" target="_blank">Fig 6b</a>) is indicated. Region A2 interacts with RNA-PolII. This interaction network recruits a repressor complex to downregulate the transcription of exon 5u <i>MB</i> transcripts under DHT treatment. Open chromatin regions are marked in black (Figure a in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.s003" target="_blank">S3 Fig</a>).</p

RNA-Seq based quantification of <i>MB</i> transcript variants in hormone treated cancer cells.

<p><b>(a)</b> Box plots of <i>MB</i> variant expression in androgen (R1881) treated LNCaP cells after 12h versus non-treated controls (n = 3; * p < 0.05). Average transcript-specific expression values are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.s005" target="_blank">S1 Table</a>. <b>(b)</b><i>MB</i> variant expression in estrogen (E2) treated MCF7 cells after 24 h versus non-treated controls (n = 7). RPKM values of RNA-Seq read counts are shown as box plots, representing transcriptional levels of the different <i>MB</i> start exons (* p < 0.05). Average transcript-specific expression values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.s005" target="_blank">S1 Table</a>.</p

Evidence for potential functional sites in candidate <i>MB</i> regulatory regions.

<p><b>(a)</b> Annotations of the hypoxia- and estrogen-responsive region A1. Large boxes indicate ChIP-Seq peaks and an RNA PolII-ChIA-PET. Small boxes and arrowheads show <i>in silico</i> predicted transcription factor binding motifs. CpGs whose methylation status has been revealed by bisulfite sequencing analyses are indicated (shown in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.s008" target="_blank">S4 Table</a>). <b>(b)</b> Annotations of the androgen-responsive region D1. The large boxes indicate AR-ChIP-Seq peaks, a DNAse I hypersensitive sites and a CTCF-ChIA-PET. Small boxes show the locations of predicted transcription factor binding motifs (AREs).</p

UCSC browser overview of ChIA-PET interactions and chromatin modifications around the <i>MB</i> exon 5u promoter.

<p><b>(a)</b> UCSC annotation of <i>MB</i> and a custom track of all human <i>MB</i> exons, according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.ref021" target="_blank">21</a>]. <b>(b)</b> UCSC browser and custom tracks with ChIA-PET coordinates. Chromosome 22 coordinates are written on top. The respective dataset specifications are listed on the right, grouped according to data showing open chromatin, histone modifications and transcription factor binding. Dark boxes in the upper section connected by thin lines reflect candidate interacting sequence regions according to ChIA-PET sequencing technique data mining. <b>(c)</b> Blue boxes indicate candidate enhancer regions, including the <i>MB</i> exon 5u promoter and DNA regions that directly interact with it based on ChIA-PET data and accumulative evidence from epigenetic marks as detailed above. The table further summarizes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.g005" target="_blank">Fig 5a</a>, Figures a in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.s003" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.s004" target="_blank">S4</a> Figs, crossmarking (X) which DNA sites show hypoxia-, androgen- and estrogen-associated chromatin modifications detected by ChIP-Seq and FAIRE-Seq.</p

Expression analysis of <i>MB</i> transcript variants in normoxic and hypoxic (1%O_2, 24 h) cancer cells.

<p><b>(a)</b> Architecture of the human <i>MB</i> gene. Boxes indicate exons, arrows mark transcription start sites (TSS) with the most active ones being labelled, based on [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.ref021" target="_blank">21</a>]. <b>(b)</b> Quantification of cDNA copy numbers in LNCaP cells, measured in triplicate assays by transcript-specific qRT-PCR and normalized on LMNB1 controls (n = 3; * p < 0.05). mRNA copy numbers per 40 ng LNCaP total RNA are shown in box plots. <b>(c)</b><i>In silico</i> quantification of <i>MB</i> transcripts by RNA-Seq analysis of MCF7 breast cancer cells (n = 2). Read counts are shown as RPKM values (* p < 0.05). Expression values are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.s005" target="_blank">S1 Table</a>.</p

UCSC browser overview of ChIA-PET interactions around the 5u <i>MB</i> promoter in MCF7 cells.

<p>Top: UCSC annotation of <i>MB</i> and a custom track of all human <i>MB</i> exons, according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.ref021" target="_blank">21</a>]. Bottom: UCSC browser and custom tracks with ChIA-PET coordinates. Dataset specifications are listed on the right. Asterisks indicate DNA regions that directly interact with the <i>MB</i> promoter. The green vertical line indicates the 5u <i>MB</i> promoter region.</p

Identification of hypoxia associated regulatory regions of the <i>MB</i> gene.

<p><b>(a)</b> UCSC browser overview of hypoxia associated chromatin modifications around the 5u <i>MB</i> promoter in MCF7 and DLD-1 cells. Top: UCSC annotation of the human <i>MB</i> gene and a custom track of all human <i>MB</i> exons, according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142662#pone.0142662.ref021" target="_blank">21</a>]. Bottom: UCSC browser and custom tracks. Dataset specifications are listed on the right. The green shattered box indicates the 5u <i>MB</i> promoter region. The red shattered boxes mark the DNA regions that directly interact with the 5u <i>MB</i> promoter site based on ChIA-PET data. The naming of the regions to match the DLRA measured constructs is written in the bottom line. <b>(b)</b> Hypoxia inducibility of the <i>MB</i> 5u promoter and interacting DNA regions in LNCaP cells. DLRAs were measured on hypoxia (1% O₂ for 72 h) and normoxia incubated cells transfected with reportergene plasmids with different DNA regions. Box plots indicate the average RLU fold change of each construct under hypoxia versus normoxia growth conditions after normalization on empty vector constructs and renilla control vectors. Standard deviations are indicated by error bars (n = 3; *** p < 0.001). <b>(c)</b> Hypoxia inducibility of the <i>MB</i> 5u promoter and interacting DNA regions in MCF7 cells. DLRAs were measured on hypoxia (1% O₂ for 72 h) and normoxia incubated cells transfected with reportergene plasmids with different DNA regions. Box plots indicate the average RLU fold change of each construct under hypoxia versus normoxia growth conditions after normalization on empty vector constructs and renilla control vectors. Standard deviations are indicated by error bars (n = 3; * p < 0.05; ** p < 0.01).</p

Inhibitory Effect of Template DNA from FFPE Tissues on PCR with <i>Pfu</i> Polymerase.

<p>A 75-bp PCR fragment within the <i>PITX2</i> gene locus was amplified using genomic template DNA from FFPE tissue (placenta) in the presence of 1 U and 2 U <i>Pfu</i> polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.</p

Successful PCR Amplification of Larger Fragments by Overcoming PCR Inhibition.

<p>PCR-amplified DNA fragments of different sizes within the <i>PITX2</i> gene locus using template DNA from FFPE tissue. The PCR was carried out using 1 µg (upper and middle panel) and 5 ng (lower panel) template DNA in the presence of 1 U and 4 U <i>Taq</i> DNA polymerase, respectively.</p