Gene expression within the amplified region.

<p>A) Expression histogram indicating the relative expression of genes mapped within the amplified region in a series of SE and EC. The stars indicate genes significantly differentially expressed between SE and EC (according to Mann Whitney U test). Most of the genes are represented by multiple specific probes; B) DNA-FISH result for SOX2 in NCCIT cells. A centromere 12 (C12) specific probe is used as control. Red dye (Cye3) shows SOX2 probe. For C12 probe green dye (FITC) is used. The blue background color is DAPi. Multiple red spots for SOX2 are detectable in each cell containing two green spots for C12, indicating SOX2 amplification. Magnification used was 630x. DNA-FISH was performed on cut tissue section with a thickness of 4 micron. This results in possible heterogeneity of the probe sizes detected. This issue was not a limitation as the purpose of this experiment was to determine the copy numbers and the size or intensity of the region. The FISH (BAC) probes were ordered at BACPAC Resources Center (BPRC) online:bacpac.chori.org. They were verified and confirmed at the department of genetics.</p

OCT3/4 and SOX2 knockdown in NCCIT.

<p>A) Percentage of positive cells for OCT3/4 and SOX2 in cells with reduced OCT3/4 and SOX2 levels compared to cells transfected with control siRNA in the NCCIT cells in three different time-points post transfection based on immunohistochemistry; The controls are set to 100 in all cases. B) Relative expression pattern of 32 genes representing targets for pluripotency and differentiation (ectoderm, mesoderm and endoderm). The expression levels are normalized based on the housekeeping gene <i>HPRT</i>; C) Percentage of living NCCIT cells with reduced level of OCT3/4 and SOX2 compared to cells transfected with control siRNA at each time point based on Trypan blue measurement; D) Percentage of positive NCCIT cells for Ki67 in untreated cells, cells transfected with control siRNA and cells with reduced levels of OCT3/4 and SOX2 at 48+12 h post transfection; E) Percentage of positive cells for Caspase 3 in untreated NCCIT cells, Cells transfected with control siRNA show a reduced level of OCT3/4 and SOX2 at all three time points after the transfection; F) FACS analysis with Propidium Iodide staining in cells transfected with control siRNA, cells transfected with SOX2 and OCT3/4 siRNA at 60 h post transfection. NCCIT cells transfected with control siRNA show the presence of 78% living cells, while cells transfected with SOX2 siRNA show 51% living cells, NCCIT cells with OCT3/4kd show 62% live cells. G) Annexin V assay results in cells with siRNA control and cells with reduced SOX2 and OCT3/4 at 60 h after transfection. In the control, percentages of living-annexin negative cells are 89.6%, while dead-annexin positive cells are 12.2%. In SOX2kd cells, the amount of living-annexin negative cells is 57.5% while the amount of dead-annexin positive cells is 17.2%. In cells transfected with OCT3/4 siRNA, living-annexin negative cells are 68.3% while dead-annexin positive cells are 12.8%. The differences between the percentages of live and dead cells in all experiments are due to using independent and various methods in order to prove apoptosis and different sensitivity of the materials and methods used. As it is demonstrated, OCT3/4 knock-down also induces apoptosis, however, this effect is not as dramatic as SOX2 knock-down. The FACS analysis were performed in triplicate.</p

Schematic representation of the hypothetical indirect regulation of SOX2 by p53 status.

<p>Wild type p53 results in induction of expression of miR-145 and miR-34a. Subsequently, miR-34a and mir-145 down-regulates pluripotency genes, including SOX2, the latter via Wnt signaling.</p

Overview mutations found and immunohistochemistry.

<p>Ex, exon; ND, not done; NA, not available; mutations indicated in bold, other samples wild type unless indicated otherwise; DG, dysgerminoma; GB, gonadoblastoma; CIS, carcinoma in situ; TE, teratoma (im: immature); YST yolk sac tumor; itSE, intratubular seminoma;</p><p>seq, sequence; LC, LightCycler; c-KIT LC, LightCycler melting curve results; case numbers in bold are bilateral cases.</p>§<p>tumor/precursor percentage below 50%.</p>‡<p>as determined by FISH on gonadal tissue.</p><p>I789I: heterozygous synonymous SNP, rs.5578615.</p><p>P567P: homozygous synonymous SNP, rs.1873778.</p

Mutational analysis of <i>SRY</i> and <i>WT1</i>.

<p>(A) wild type (upper panel, control) and mutated sequence (lower panel, patient) of <i>SRY</i>. (B) schematic representation of the SRY protein. The K128R mutation resides in the HMG domain, just before the cNLS. (C) <i>In vitro</i> luciferase assays of SRY-WT (wild-type) and SRY-K128R (mutant) in HEK293T cell line. Cells were co-transfected with TESCO-E1b-<i>Luc</i>, SF1 and WT or mutant SRY to assess for activation of TESCO. The mean percentages of fold change of luciferase activity of TESCO-E1b-<i>luc</i> over the empty vector, relative to WT SRY levels from six independent assays (each performed in triplicate) are shown. Error bars represent standard error of the mean (SEM). (D) pcDNA3-FLAG-SRY wild-type (WT, 2 µg) or pcDNA3-FLAG-SRY mutant (K128R, 2 µg) were transiently transfected into HEK293T cells using Fugene 6. Exogenous SRY (WT or K128R) expression was detected using a FLAG antibody and a green fluorescent Alexa-488 dye coupled secondary antibody. Nuclei were stained with 4′,6-diamino-2-phenylindole (DAPI). Both wild type and mutant SRY show strong nuclear staining. (E) SRY fluorescence was quantified as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040858#pone.0040858-Argentaro1" target="_blank">[35]</a>. Nuclear accumulation of SRY (WT or K128R) expressed as fluorescence in the nucleus over that in the cytoplasm (Fn/c) were background fluorescence has been subtracted. Measurements represent the average of 3 independent transfections. Results are relative to WT transfected cells (Fn/c given value of 100%). The number of cells analysed is n = 111 (WT) and n = 121 (K128R). Error bars represent the standard error of mean values. Two-tail t-Test of unpaired sample means was performed between WT transfected cells and mutant transfected cells and showed no significant differences. P = 0.49. (F) mutated sequence (upper panel, patient) and wild type sequence (lower panel, control) of <i>WT1</i>, showing the heterozygous +4C&gt;T change.</p

Immunohistochemical staining of the left GB lesion.

<p>(A) representative hematoxylin and eosin (HE) staining. The germ cells present in the GB stain positive for OCT3/4 (B, brown staining), TSPY (C, red staining), and SCF (D, red staining). Supportive cells in the GB stain positive for FOXL2 (E, brown staining), while SOX9 (F) is negative. All slides are counterstained with hematoxylin. Magnification 100x for all.</p

Localization and expression of LRRC50.

<p>LRRC50 antibody ab75163 (reproducible with alternative LRRC50 antibodies) shows a dynamic, cell cycle dependent, distribution in RPE-hTERT cells (representative for other tested mammalian cell lines). Panels represent LRRC50 (green) at various stages, counterstained with DAPI (blue) and acetylated-α-tubulin (Ac-tub, red). Optical sections in A–C are 3 µm. (A) In ciliated serum-starved cells, LRRC50 localizes to the peripheral centrosome/basal body region dorsal of the axoneme (red). Scale bar 10 µm. (B) In mitotic cells, LRRC50 remains associated with the duplicated centrosomes, as indicated with γ-tubulin (red). Inserts b1,2 demonstrate a peri-centrosomal localisation. Scale bar 2 µm. (C) Temporal localization to the midbody in cytokinesis. Scale bar 10 µm. (D) During mitosis LRRC50 dynamically associates with condensed chromosomes (extensively described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003384#pgen.1003384.s005" target="_blank">Figure S5B</a>). Counterstain CREST (red) marks kinetochores. Image is a maximum intensity projection of deconvoluted stacks. Scale bar 2 µm. (E) Dynamic <i>LRRC50</i> mRNA expression (correlating with dynamic localization) with error bars as standard deviation. T47D cells synchronized at the G1/S transition with thymidine show a strong <i>LRRC50</i> transcript up-regulation upon release, specifically in the cell population entering early S-phase (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003384#pgen.1003384.s006" target="_blank">Figure S6A</a>). Intriguingly; although the protein remains stable during mitosis (shown in D) the transcript is rapidly down-regulated, and restored to basal levels. (F) Expression profiling of <i>LRRC50</i> mRNA expression with error bars of standard deviation in a library of mouse cDNA tissues normalized to full mouse reference pool. Testis expression shown in red as the expression level (>26.000%) strongly exceeds the normalized value.</p

Characterization of <i>lrrc50^Hu255h</i> zebrafish TGCT suggests analogy to human seminoma.

<p>(A–D) Histological characterization of wild-type testis (left panels) and <i>lrrc50^H255h</i> tumors (right panels). Two magnifications shown A, B (largest in insert) all scale bars; 50 µm. The characterization indicates the presence of predominantly early germ cells and loss of differentiated germ cells in the tumors. (A) Morphological tissue analysis of toluidine blue stained sections indicates the presence of all stages of spermatogenesis in normal tissue (extensively described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003384#pgen.1003384.s003" target="_blank">Figure S3A</a>), and shows a dramatic loss of differentiated germ cells in the tumor. (B) IHC characterization with proliferation marker α-<i>phospho</i>-HistoneH3 (pH3) shows synchronously dividing cell-clusters in normal tissue, indicative of differentiated germ cells. Early SPG is the only germ cell that can divide as a single cell, and the tumors show mostly single proliferating cells. Increased pH3 staining suggests the tumor tissue is highly proliferative (quantified in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003384#pgen.1003384.s004" target="_blank">Figure S4A</a>). (C) IHC using α-Ziwi (strong cytoplasmic expression). In normal tissue, Ziwi expression is restricted to early SPG and gradually and diffusely lost differentiated germ cells. With the exception of somatic tissue, the tumors are almost completely composed of early SPG. (D) IHC with meiosis marker α-γ-H2Ax shows normal tissue that is composed of various stages of differentiation, whereas these are predominantly absent in tumor tissue. (E) Chromatograms of WT zebrafish, heterozygote <i>lrrc50^Hu255h</i> and <i>lrrc50^Hu255h</i> tumors. We observe a loss of the remaining wild-type allele c.263T>A/p.Lys88* in 44.4% of the tumors (LOH).</p

Human TGCT samples.

<p>Genotypic analysis was performed on human TGCT samples (n = 51); a total of 30 seminomas (SE) and five spermatocytic seminomas (SS) were initially analyzed. These samples were expanded with 15 fTGCT samples, including eight seminomas, which were isolated from patients known to have a familial background of seminomas. Non-SE = non-seminoma, EC = embryonic carcinoma, (im)Te = (immature) teratoma, YS = yolk sac tumor, Ch = choriocarcinoma. Patients carrying alleles identified in this study are indicated.</p

Heterozygous <i>lrrc50^Hu255h</i> zebrafish are predisposed to testicular tumor formation.

<p>(A) Incidences of tumors extracted from randomly selected male controls derived from ENU-mutagenesis (n = 104) and heterozygote male <i>lrrc50^Hu255h</i> (n = 30) zebrafish between 24–44 months of age are summarized in pie charts. Tumor formation presented as both TGCT and non-TGCT (sporadic tumors in somatic tissues) in the <i>lrrc50^Hu255h</i> cohort (90%) is significantly elevated from TGCT (16.3%) formation in the controls (<i>P</i><0.0001). No alternative tumor types were noticeable in the control group. (B) An age-matched wild-type zebrafish compared to a TGCT bearing <i>lrrc50+/−</i> zebrafish (skin around tumor removed; merge of three images). (C) Wild-type testes are composed of two tubular arms forming the paired gonad. The tissue architecture is severely disrupted in the tumor.</p