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