Relationship between clinicopathological parameters and <i>BCAR4</i> mRNA expression in primary BCs.

<p>^aFisher’s exact test,</p><p>^bChi Square test for trends,</p><p>^c Fisher’s exact test (0, 1+, 2+/Fish-negative <i>versus</i> 3+).</p><p>Relationship between clinicopathological parameters and <i>BCAR4</i> mRNA expression in primary BCs.</p

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

<i>BCAR4</i> mRNA expression, as determined by (q)RT-PCR in primary, pre-treatment BCs.

<p>Cases are ordered by <i>BCAR4</i> mRNA expression level (A), or by molecular subtype (B). Error bars represent SEM.</p

Characteristics of primary, pre-treatment BCs.

<p>Characteristics of primary, pre-treatment BCs.</p

Lapatinib counteracts <i>BCAR4</i>-induced cell proliferation.

<p>(A) Expression of ERBB2 and EGFR in human BC cell lines, as detected by immunohistochemistry using the clinically validated antibodies 4B5 (ERBB2) and 2.1E1 (EGFR). (B) Expression of ERBB2 and EGFR in human BC cell lines, as detected by western blot using the same immunological reagents. BT20 and MDA-MB-468 BC cells served as positive controls for EGFR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136845#pone.0136845.ref029" target="_blank">29</a>]. (C) IPH-926 cells were exposed to siRNAs against <i>EGFR</i> (siEGFR), <i>ERBB2</i> (siERBB2), <i>ERBB3</i> (ERBB3), <i>ERBB4</i> (ERBB4) or to the transfection mix (TF-mix) reagents only and cell proliferation was measured with the WST-1 assay. Averages of minimal 6 replicates and Error bars representing the SEM are presented. (D) Cells were exposed to various concentrations of lapatinib for five days and cell proliferation was determined with the WST-1 assay. Data are presented as relative proliferation normalized to untreated controls and error bars represent SEM.</p

Detection of BCAR4 protein expression with a polyclonal anti-BCAR4 antibody (C78-I97).

<p>(A) BCAR4 protein expression in ZR-75-1 BC cells retrovirally transduced with various expression constructs as indicated. V: ZR-75-1 cells transduced with empty vector, B1: BCAR1, B3: BCAR3, B4: BCAR4 E: EGFR, G: EGFP, GB4: EGFP-BCAR4. (B) Western blot analysis of Flag-tagged BCAR4 immunoprecipitated with C78-97 and detected with anti-Flag antibodies. (C) BCAR4 protein expression in human placenta tissue. (D) Western blot analysis of BCAR4 protein expression by immunoprecipitation in human BC cell lines. (E) Human BC cell lines were exposed to siRNAs directed against <i>BCAR4</i> (siBCAR4, average of three individual siRNAs), <i>EGFR</i> (siEGFR) or to the transfection mix (TF-mix) reagents only. Subsequently, cell proliferation was measured with the WST-1 proliferation assay. Averages of minimal 6 replicates. Error bars represent the SEM.</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

Characteristics of included studies.

<p>Characteristics of included studies.</p

Forest plot of studies investigating p53 as a predictor of progression.

<p>Twelve studies were included.</p