IKAP is expressed in oligodendrocytes in vivo.

<p>Sagittal mouse brain cryosection at the level of dentate gyrus immunostained for IKAP (green) and the oligodendrocyte marker GALC (red), counterstained with DAPI (blue). (<b>A</b>) Arrow shows an oligodendrocyte, which stains for GALC and IKAP simultaneously (yellow). (<b>B</b>) IKAP expression (green channel), (<b>C</b>) GALC expression (red channel), and (<b>D</b>) DAPI staining (blue channel).</p

IKAP Deficiency in an FD Mouse Model and in Oligodendrocyte Precursor Cells Results in Downregulation of Genes Involved in Oligodendrocyte Differentiation and Myelin Formation

<div><p>The splice site mutation in the <i>IKBKAP</i> gene coding for IKAP protein leads to the tissue-specific skipping of exon 20, with concomitant reduction in IKAP protein production. This causes the neurodevelopmental, autosomal-recessive genetic disorder - Familial Dysautonomia (FD). The molecular hallmark of FD is the severe reduction of IKAP protein in the nervous system that is believed to be the main reason for the devastating symptoms of this disease. Our recent studies showed that in the brain of two FD patients, genes linked to oligodendrocyte differentiation and/or myelin formation are significantly downregulated, implicating IKAP in the process of myelination. However, due to the scarcity of FD patient tissues, these results awaited further validation in other models. Recently, two FD mouse models that faithfully recapitulate FD were generated, with two types of mutations resulting in severely low levels of IKAP expression. Here we demonstrate that IKAP deficiency in these FD mouse models affects a similar set of genes as in FD patients' brains. In addition, we identified two new IKAP target genes involved in oligodendrocyte cells differentiation and myelination, further underscoring the essential role of IKAP in this process. We also provide proof that IKAP expression is needed cell-autonomously for the regulation of expression of genes involved in myelin formation since knockdown of IKAP in the Oli-neu oligodendrocyte precursor cell line results in similar deficiencies. Further analyses of these two experimental models will compensate for the lack of human postmortem tissues and will advance our understanding of the role of IKAP in myelination and the disease pathology.</p></div

IKAP knockdown in Oli-neu cells results in reduced expression of myelin-related genes.

<p>Quantitative real-time PCR (qPCR) analysis of APOD, EDG2, Ermin, GTX, KLK6, MAG, MAL, MBP, PLP1, PP1R14A, SST, TMEM10, Transferrin, and TTYH2 in RNA from control (ShSCR) and Ikbkap knockdown (ShIKAP) Oli-neu cells. Analysis was repeated 2 to 3 times for each gene. Expression levels were normalized over internal control (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094612#s4" target="_blank">Materials and Methods</a>) and are expressed as percentage of CTRL. Data are represented as Mean ±SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.</p

Reduced expression of IKAP target genes in FD mouse models.

<p>Quantitative real-time PCR (qPCR) analysis of APOD, EDG2, Ermin, GTX, KLK6, MAG, MAL, MBP, PLP1, PP1R14A, TMEM10, Transferrin, and TTYH2 in brain RNA from control (CTRL, n = 8) and FD (n = 7). Analysis was repeated 2 to 3 times for each gene. Expression levels were normalized over internal control (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094612#s4" target="_blank">Materials and Methods</a>) and are expressed as percentage of CTRL. Data are represented as Mean ±SD. **P<0.01, ***P<0.001, ****P<0.0001.</p

IKAP protein levels are highly reduced in FD mutant mice and after lentiviral ShIKAP administration in Oli-neu cells.

<p>(<b>A</b>) Western blot analyses of total protein lysates from brain cortex of control (N1, N2, N3) and age-matched FD (FD1, FD2, FD3) littermates. Upper panel shows detection of IKAP with the polyclonal anti-IKAP antibody (AnaSpec), and lower panel shows anti-β-actin for loading control. Note that IKAP protein expression is highly reduced in FD1, FD2, and FD3, relative to controls. Quantitative analysis of the Western blot was performed using Image J software. IKAP expression levels over β-actin levels are presented. (<b>B</b>) Western blot analyses of total protein lysates from control scrambled (ShSCR) and <i>IKAP</i> knockdown (ShIKAP) Oli-neu cells. Upper panel shows detection of IKAP with the polyclonal anti-IKAP antibody (AnaSpec), and lower panel shows anti-β-actin for loading control. Note that IKAP is significantly expressed in ShSCR Oli-neu cells. Quantitative analysis of the Western blot was performed using Image J software. IKAP expression levels over β-actin levels and are presented.</p

Cols bleus : hebdomadaire de la Marine française

22 mars 19971997/03/22 (N2389)-1997/03/22

Cols bleus : hebdomadaire de la Marine française

17 mai 19801980/05/17 (N1614)-1980/05/17

Additional file 10: Table S9. of The signature of liver cancer in immune cells DNA methylation

Multifactorial ANOVA analysis of 350 CGs. No interaction detected between group (HCC) and sex and age as independent variables with CG methylation as a dependent variable. (CSV 31 kb

Additional file 6: Table S6. of The signature of liver cancer in immune cells DNA methylation

Annotated non-redundant list of 350CGs and 369 CGs that are differentially methylated between stages of HCC and healthy controls. (CSV 41 kb

Additional file 16: Table S15. of The signature of liver cancer in immune cells DNA methylation

Descriptive statistics for (Fig. 6a). (XLS 87 kb