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Chromosomal localization and relative cloning frequencies of cDNAs derived from transcribed HERV-E4-1 loci in malignant and non-malignant tissue of patient no. 2.

1<p>aliases according to HUGO Gene Nomenclature Committee are in parentheses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049341#pone.0049341-Mayer1" target="_blank">[90]</a>.</p>2<p>Chromosomal localization of MOP-PCR amplicons according to the hg18/March 2006 human reference genome sequence as given by the UCSC Genome Browser.</p>3<p>HERV-Ec8 was mapped within a duplicated genome region, with both HERV-Ec8 sequences displaying only 20 nt differences along 8 kb. Assignment to one or the other locus is therefore not possible due to high sequence similarity.</p>4<p>Total number of sequenced cDNA clones.</p><p>Abbreviations: no., number; chr, chromosome.</p

Relative transcript levels of selected HERV elements in patient samples.

<p>(A) Relative positions (not drawn to scale) of siRNAs (red bars) in the HERV-E4-1 5′-LTR. Location and sizes of qRT-PCR and MOP-PCR products (RetroArray) are shown. Given amplicon sizes exclude PCR primers. (B) Quantitative analysis of HERV-E4-1, HERV-K(HML-6) and HERV-T(S71-TK1) transcript levels in cDNA samples derived from 18 non-malignant urothelium (N) and 35 urothelial carcinoma (T) tissue specimen, including 16 paired tissue samples. HERV subgroup-specific <i>pol</i> primers for HERV-E4-1 and HERV-T(S71-TK1), and degenerated <i>pol</i> primers for HERV-K(HML-6) were used. Relative transcript levels were quantified according to Pfaffl <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049341#pone.0049341-Pfaffl1" target="_blank">[52]</a>. All qRT-PCR values were normalized to GAPDH levels. HERV-E4-1 mean expression of N vs. T was significantly different (p = 0.037; Student’s <i>t</i>-test). (C) Relative cloning frequencies of HERV-E4-1-related cDNAs (as shown in Tab. 3) were combined with the respective HERV-E4-1 qRT-PCR data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049341#pone-0049341-g002" target="_blank">Figure 2B</a>) to illustrate the differential activities of transcriptionally active HERV-E4-1 loci in malignant (T) and non-malignant (N) tissues of patient no. 2.</p

Comparative analysis of HERV-Ec1 <i>gag</i> and PLA2G4A transcription in patients with urothelial carcinoma.

<p>QRT-PCR assays were performed on DNA-free RNA samples obtained from patients with urothelial carcinoma (n = 11). Due to lack of material, additional patients were used not shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049341#pone-0049341-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049341#pone-0049341-g002" target="_blank">2</a>. For measurement of HERV-Ec1 transcripts locus-specific primers derived from the <i>gag</i> region of HERV-Ec1 were utilized. Transcript levels of PLA2G4A were analyzed using a gene specific primer set. Relative transcription levels were normalized to G6PD transcript levels and represent the mean value of triplicate qRT-PCR assays. The numbers on the Y-axis show the fold change of transcription in urothelial carcinoma with respect to corresponding mean value of non-malignant tissue samples (value = 1, depicted by red horizontal line). Thus, values >1 denote increases, values <1 correspond to decreases in transcript levels.</p

Effect of aza-dC on transcription of PLA2G4A in UROtsa cells.

<p>UROtsa cells were incubated with 5 µM 5-aza-2-deoxycytidine (aza-dC) for 48 h and analyzed by qRT-PCR in comparison to untreated cells. QRT-PCR assays were performed on DNA-free RNA samples derived from two independent experiments. Primer pairs derived from <i>pol</i> and <i>gag</i> regions were used. While HERV-E4-1 <i>pol</i>-targeting primers overlap the capture probes of the RetroArray and may amplify several HERV-E4-1 loci, the HERV-Ec1 <i>gag</i> primers are specific for the HERV-Ec1 provirus located within intron 7 of the PLA2G4A gene. Transcript levels of PLA2G4A were analyzed using gene-specific primers. Relative transcription levels were normalized to G6PD levels and represent the mean value of six qRT-PCR assays. Numbers on the Y-axis show the fold change of transcription. (*) An almost complete downregulation of HERV-Ec1 <i>gag</i> transcription (0.00027±0.00017%) was observed.</p

HERV transcriptional profile of the human urothelium¹.

1<p>Numbers of samples positive for the respective HERV out of 13 paired (malignant and non-malignant) patient samples. Samples were considered positive, when their corresponding signals exceeded the calculated cut-off value as described in Materials & Methods. Incidences are given by absolute numbers of samples and do not represent any quantitative assessment of HERV transcript levels.</p>2<p>UROtsa cells were used as model system of the normal human urothelium.</p>3<p>Tumor cell lines under investigation: RT112, T24/83, UMUC-3, HT1197.</p>4<p>HERV subgroups selected for quantification by qRT-PCR.</p>5<p>HERV subgroups that represent the urothelium-specific core HERV signature.</p

Syncytin-1 protein expression is found in MF skin lesions but not in lichen planus.

<p>A) Morphologically malignant lymphocytes within Pautrier’s microabscesses (invading the epidermis) stained positive (red arrow) for Syncytin-1 in immunohistochemistry, DAB 40x B) same sample as in a) but with NovaRED as a chromogen, 100x, C) same sample as in A) without primary antibody, DAB 40x, D) Lichen ruber planus sample with no Syncytin-1 expression albeit abundant numbers of inflammatory T cells, NovaRED 20x, E) in folliculotropic MF, Syncytin-1-positive lymphocytes protruding in the hair follicle, 20x and F) human placenta as a positive control, 20x. Scale bar is 20µm.</p

Retroarray analysis of HERV transcriptional activity in MF.

<p>HERV activity profiles representing pairs of lesion (A) and non-malignant (B) skin tissue specimens (digitally aligned). Each sample pair (n=17) was derived from an individual patient (MF 1-12, psoriasis 13-17). HERV subgroups representing a skin-specific core transcription profile (HERV-E, HERV-F, HERV-W, ERV-9, HERV-K(HML-4)) are emphasized with red letters. A panel of housekeeping genes (Ubiquitin, GAPDH, RPL19, β-Actin, HPRT) served as internal controls (for detailed information on methodology, see [77,79]). Each positive spot on the microarray may represent multiple HERV loci of one subgroup of multicopy HERV elements with sufficient sequence similarity that individual elements cannot be distinguished. Weak signals may be unrecognizable in the printed figure.</p

HERV transcription activity in patient samples by RetroArray analysis.

<p>HERV activity profiles representing pairs of malignant (T) and non-malignant (N) urothelial tissue specimen, each pair derived from the same patient (n = 13) were digitally aligned. Below, the HERV signatures of four urothelial cancer cell lines (RT112, T24/83, UMUC-3, HT1197) and of the non-malignant urothelial cell line UROtsa are shown. HERV subgroups representing the urothelial core profile are emphasized with red letters. The housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT) served as internal control. For detailed information about the identity of targets and capture probes, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049341#pone.0049341.s002" target="_blank">Table S1</a> and references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049341#pone.0049341-Seifarth1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049341#pone.0049341-Seifarth3" target="_blank">[41]</a>. Each positive spot on the microarray represents multiple HERV loci assigned to one subgroup of multicopy elements with sufficient sequence similarity so that individual elements cannot be distinguished. Although false color mapping was used for improved image visualization, weak signals may be unrecognizable in the figure. QRT-PCR was performed for HERV-E4-1, HERV-T(S71-TK1) and HERV-K(HML-6), as depicted by red boxes.</p

Relative expression of <i>ERVWE1</i> mRNA in MF skin tissue.

<p><i>ERVWE1</i> expression was detected in 3/7 MF lesions analysed by Taqman qPCR. Patients 3 and 7 were also analysed, but the MF lesions showed no <i>ERVWE1</i> expression (Figure S1). Patients 7 and 18 showed expression also in the clinically healthy (H), non-lesional skin samples (the data of patient 7 in Figure S1). <i>GAPDH</i> served as internal standard and was expressed in every sample (see Methods for more details). MF patients 18 and 19 were collected afterwards and were therefore not included in other experiments. See detailed amplification curves in Figure S1.</p