Analysis of the Illumina HumanGW 6 BeadChip whole genome expression array.

<p>(<b>A</b>) Hierarchical clustering by Pvclust by of the significantly expressed genes in HEK293-cells transfected with siRNAs for the FET mRNAs (siFUS, siEWS, siTAF15, siFUS+siEWS+siFUS) or an unspecific control siRNA (control + and control +++). The control + is control sample for the individual FET siRNA transfections, and the control +++ for the siFUS+siEWS+siTAF15 sample, to ensure the comparisons of equal siRNA concentrations. P-values (%) are shown on the edges of the clustering, Approximately Unbiased (AU) p-values in red, and Bootstrap Probability (BP) values in green. Clusters with an AU p-value greater than 95% are highlighted by a red rectangle. (<b>B</b>) The distribution of differentially expressed genes (DEGs) in the siFUS (yellow), siEWS (blue), and siTAF15 (light red) transfected cells. The up-regulated number of genes is shown in red and down-regulated in green. The number of common DEGs is shown in the overlapping parts of the circles. (<b>C</b>) DEGs in the siFUS, siEWS, and siTAF15 transfected cells. The common DEGs were compared to the DEGs in the siFUS+siEWS+siTAF15 transfected cells. In total 637 DEGs are identified in the siFUS+siEWS+siTAF15 cells, of those are 512 unique genes (grey). 33 of the 319 common DEGs in siEWS transfected cells were found (black), 38 of 226 in FUS (red), 29 of 221 in TAF15 (green), 10 of 38 in FUS and EWS (yellow), 2 of 29 in EWS and TAF15 (dark blue), 5 of 22 in FUS and TAF15 (pink), and 8 of 21 in FUS and EWS and TAF15 were found (blue).</p

Western blot of co-immunoprecipitation of the FLAG-TIA1 and FLAG-TIAR proteins shows no significant binding to FUS, EWS, or TAF15.

<p>(<b>A</b>) Co-immunoprecipitation conducted without RNase A. The used FLAG cell line is shown above the blot. “Emp” is HEK293-cells without FLAG vector used as control. Unstressed control cells are marked (-), and stressed arsenite treated cells (Ars). (*) marks an unidentified background protein, and HC and LC is heavy and light chain, respectively, from the used mouse antibodies. HuR is used as control protein for an intact RNA dependent interaction to TIA1 and TIAR. (<b>B</b>) The co-immunoprecipitation conducted with RNase A. (*) marks an unidentified background protein, and HC and LC is heavy and light chain respectively from the used mouse antibodies.</p

Immunostainings of the FET and TIA1 proteins in HEK293 cells after siRNA-mediated gene knock-down of the FET proteins together with arsenite induced stress.

<p>Control cells were transfected with an equal amount of siRNA with an unspecific sequence. (<b>A</b>) TIA1 and FUS immunostaining. The nuclei are counterstained by DAPI. (<b>B</b>) TIA1 and TAF15 immunostaining. The nuclei are counterstained by DAPI (<b>C</b>) TIA1 and EWS immunostaining. The nuclei are counterstained by DAPI.</p

FET-protein reduction is not affecting ROS.

<p>(<b>A</b>) HEK293 cells were transfected with siRNAs targeted against the FUS, EWS, or TAF15 mRNAs, or with an unspecific siRNA as control. Cells were also transfected with a combination of all three FET siRNAs (300 nM total concentration) or unspecific siRNA at concentration of 300 nM (lower panel). The level of ROS in the cells was measured and evaluated by microscopy. Untransfected cells treated with TBHP is a positive control of oxidative stress (upper panel left), and untransfected cells without TBHP treatment is a negative control of oxidative stress (upper panel right). (<b>B</b>) HEK293 cells were transfected with a combination of all three FET siRNAs (300 nM total) or with an unspecific siRNA as control. Cells were subsequently treated with TBHP to induce oxidative stress (left columns), and the level of ROS was measured and evaluated by microscopy and compared to unstressed cells (right columns). Untransfected cells treated with TBHP is a positive control of oxidative stress (upper panel left), and untransfected cells without TBHP treatment is a negative control of oxidative stress (upper panel right).</p

Verification of the gene expression array analysis.

<p>(<b>A</b>) RT-qPCR analysis of siFUS, siEWS, siTAF15, and siFUS+siEWS+siTAF15 transfected HEK293 cells. 13 candidate genes which expression was altered in the array analysis were chosen. RT-qPCR was performed in triplicates, and the gene expression was normalized to the expression of the housekeeping gene GAPDH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046251#pone.0046251-Thomsen1" target="_blank">[45]</a>. The expression level as measured by RT-qPCR is shown in black, and the measured level in the array is shown in white. Standard deviations from the technical triplicates are shown. (<b>B</b>) FUS, EWS, and TAF15 protein expression in the siFUS, siEWS, and siTAF15 transfected HEK293 cells used in the expression analysis. Control cells are transfected with an equal amount of unspecific siRNA. The protein expression of the ubiquitously expressed protein hnRNPC1/C2 (heterogeneous nuclear ribonucleoprotein C (C1/C2)) is shown as loading control. (<b>C</b>) FUS, EWS, and TAF15 protein expression in the siFUS+siEWS+siTAF15 transfected HEK293 cells used in the expression analysis. Control cells are transfected with an equal amount of unspecific siRNA. The protein expression of hnRNPC1/C2 is shown as loading control.</p

RT-qPCR of the FET mRNA expression in HEK293 and SH-SY5Y cells after oxidative stress.

<p>The FET mRNA levels were normalized to the expression of GAPDH and quantified to the FET mRNA level in unstressed cells according to the X₀-method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046251#pone.0046251-Thomsen1" target="_blank">[45]</a>. (<b>A</b>) Arsenite induced cellular stress. *, P<0.001. **, P<0.01. (<b>B</b>) H₂O₂ induced cellular stress. *, P<0.005. **, P<0.01.</p

El Distrito Universitario : Semanario de 1ª Enseñanza: Año XVI Número 834 - 1918 agosto 2

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Triploidy—Observations in 154 Diandric Cases

<div><p>Hydatidiform moles (HMs) are abnormal human pregnancies with vesicular chorionic villi, imposing two clinical challenges; miscarriage and a risk of gestational trophoblastic neoplasia (GTN). The parental type of most HMs are either diandric diploid (PP) or diandric triploid (PPM). We consecutively collected 154 triploid or near-triploid samples from conceptuses with vesicular chorionic villi. We used analysis of DNA markers and/or methylation sensitive-MLPA and collected data from registries and patients records. We performed whole genome SNP analysis of one case of twinning (PP+PM).In all 154 triploids or near-triploids we found two different paternal contributions to the genome (P1P2M). The ratios between the sex chromosomal constitutions XXX, XXY, and XYY were 5.7: 6.9: 1.0. No cases of GTN were observed. Our results corroborate that all triploid human conceptuses with vesicular chorionic villi have the parental type P1P2M. The sex chromosomal ratios suggest approximately equal frequencies of meiosis I and meiosis II errors with selection against the XYY conceptuses or a combination of dispermy, non-disjunction in meiosis I and meiosis II and selection against XYY conceptuses. Although single cases of GTN after a triploid HM have been reported, the results of this study combined with data from previous prospective studies estimate the risk of GTN after a triploid mole to 0% (95% CI: 0–1,4%).</p></div

Parental types in triploid conceptuses with molar phenotype.

<p>Parental types in triploid conceptuses with molar phenotype.</p

reproduced [7]. Possible fertilizations, endoreduplications, and abnormal cell divisions in mosaic hydatidiform moles (HMs), and twin gestations including an HM.

<p>(a) Fertilisation by two sperms; one giving rise to the paternal genome set in the diploid biparental cell population, the other giving rise to both genome sets in the diploid androgenetic cell population via endoreduplication. (b) Fertilisation by two sperms; one giving rise to one of the paternal genome sets in the diploid androgenetic cell population, the other contributing one genome set to both cell populations via endoreduplication. (c) Fertilisation by one sperm that via two endoreduplications gives rise to three identical paternal genome sets, of which two constitute the genome of the diploid androgenetic cell population and one is the paternal genome set in the diploid biparental cell population.</p