Analysis of Tau phosphorylation in 4RTau cell lines by 2D electrophoresis.

<p>A) Bi-dimensional analysis of <i>WT</i> and Mutated <i>P301S</i> or <i>S305N</i> 4RTau. Differentiated cells treated with tetracycline for 48 hours were subjected to 2D analysis and immunoblotted with Tau Exon10 antibody (4RTau specific). Alignments of immunoblots were performed using unspecific products revealed by secondary antibody. Different molecular weight (MW) levels (levels1–3: L1–L3) of Tau isovariants are represented by short dashed lines and pI indicators (obtained with an internal IEF control) by long and short mixed dashed lines. B) Bi-dimensional analysis of <i>WT</i> and Mutated 4RTau after <i>in vitro</i> Tau dephosphorylation followed by immunoblotting with Tau exon10 antibody. C) Immunoblot analysis of <i>in vitro</i> dephosphorylated <i>WT</i> and mutated (<i>P301S</i> and <i>S305N</i>) 4RTau. Differentiated cells treated with tetracycline for 48 hours were subjected (+) or not (−), to an <i>in vitro</i> dephosphorylation by λ-phosphatase and immunoblotted by phosphorylation dependent antibody. Total amount of Tau proteins loaded is visualized with TauCter 1902 antibody (Total Tau) and β-actin was used as loading control.</p

Analysis of Tau aggregation by electronic microscopy and Gold-immunolabeling.

<p>Lysates from differentiated Mock and 4RTau cell lines, treated with tetracycline for 48 hours, were subjected to immunogold labelling with TauCter 1902 (Total Tau) antibody and revealed by colloidal gold labelled secondary antibody. Images were obtained at high magnification: 20 000×. “Clustering” of Tau immunoreactivity is indicated by black arrows.</p

Analysis of overall Tau phosphorylation in THY-Tau 22 transgenic mice.

<p>Comparison of 2D analysis of human exogenous Tau in 3-weeks old Thy-Tau22 trangenic mice and mutated <i>P301S</i> 4RTau from SY5Y Cells. Alignments of immunoblots were performed using unspecific products revealed by secondary antibody. Different molecular weight (MW) levels (L1–L3) of Tau isovariants are represented by short dashed lines and pI indicators (obtained with an internal IEF control) by long and short mixed dashed lines. Exogenous Tau in mice was probed by an anti-human Tau antibody and exogenous <i>P301S</i> 4RTau in SY5Y cells was visualized by Tau-Exon10 antibody.</p

Antibodies used in immunoblotting studies.

*<p>Blocking: Tris-buffered saline, pH 8, 0.05% Tween 20+5% skim Milk.</p

Analysis of transgenes expression in 4RTau cell lines.

<p>A) Lysates from Mock and 4RTau cells, treated or not (0) with tetracycline for 24 to 48 hours were immunolabeled with TauCter 1902 (Total Tau), and β-actin as loading control. B) Quantification of Tau expression levels in 4RTau cell lines: Ratios of densitometric values of TauCter1902/β-actin immunoreactivities are presented. Ratios are normalized to those obtained from <i>WT</i> 4RTau cells after 48 hours tetracycline (arbitrary value = 1).</p

Differences in alternative splicing events (ASEs) when normal adult muscle cell lines are compared to normal embryonic muscle cell lines.

<p>(<b>A–C</b>) <b>A.</b> Histograms representing the four ASEs (<i>ABCB8</i>, <i>C10orf58</i>, <i>ACTN1</i>, <i>ENO3</i>) that are differentially spliced when normal embryonic cell lines (white bars) are compared to normal adult cell lines (black bars). <b>B.</b> Venn diagram representing hits when the three embryonic cell line categories (ST-750, ST-1200, and ST-3500) were compared to normal fetal cell lines. <b>C.</b> Histograms representing Ψ values in the normal fetal (white bars), ST-750 (light grey bars), ST-1200 (dark grey bars) and ST-3500 cells (black bars). Only the top 27 of the 50 splicing alterations seen in ST-3500 (<i>q</i> ≤ 0.05) are shown which also include all ST-750 (†) and ST-1200 (*) hits (respective <i>q</i>≤0.05) relative to normal fetal cells.</p

Role of MBNL1 and RBFOX1 in splicing regulation.

<p>(<b>A–C</b>) <b>A.</b> Venn diagram representing the overlap of hits obtained by knocking down MBNL1 and RBFOX1 in the HFN embryonic muscle cell line. In panels <b>B</b> and <b>C</b>, Venn diagrams are presented to illustrate events coregulated by MBNL1 and RBFOX1 that are mis-spliced in embryonic DM1 lines or and DM1 adult samples. The number and identity of the ASEs in each category are indicated. Gene names in bold indicate that the splicing shift for those ASEs occur in the reverse direction to the DM1 mis-splice.</p

Alternative splicing events co-regulated by MBNL1 and RBFOX1.

<p>Genes in bold are mis-spliced in DM1 tissues.</p><p>Alternative splicing events co-regulated by MBNL1 and RBFOX1.</p

Splicing defects in DM1 patient tissues.

<p>(<b>A–B</b>) <b>A.</b> Ψ values for ten misspliced ASEs are represented as histograms for 4 adult controls (white bars) and 5 DM1 patients (black bars). Error bars represent standard deviations for each ASE. Hits were defined as changes displaying <i>q</i> values <0.05 and |ΔΨ| >5%. <b>B.</b> Sample UCSC Genome Browser (<a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>) adaptation showing the chromosome 19 region harboring human insulin receptor, INSR. Top image shows reported full-length RefSeq transcripts, the targeted ASE is boxed and shown in detail in the bottom image. The positions and names of the primers used for mRNA amplification by RT-PCR are shown above the transcripts. Links to transcript maps and primer positions for all human ASEs studied here can be found at <a href="http://palace.lgfus.ca/data/related/2073/odgene_/" target="_blank">http://palace.lgfus.ca/data/related/2073/odgene_/</a>.</p

Splicing defects in a mouse strain expressing CUG repeats.

<p>Total RNA from muscle tissues of transgenic C57BL6 mice expressing 600 and 1200 CUG-repeats were screened for alternative splicing defects. We interrogated 172 ASEs in genes reported to be susceptible to changes in HSA^LR and MBNL knockout mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107324#pone.0107324-Du1" target="_blank">[14]</a>. Using a false discovery rate threshold (<i>q-</i>value) of 0.05 and |ΔΨ| greater than 5 percentage points, we identified 24 ASEs in CUG1200 (black bars) that are significantly different from WT (white bars). Changes that were also significant in CUG600 (grey bars) are indicated with an asterisk. Results are presented in histograms by order of significance based on <i>q</i>-values.</p