Upregulation of <i>App</i>, <i>Adam10</i> and <i>Adam17</i> mRNAs in the dentate gyrus at 7 days after entorhinal denervation.

<p>Expression levels of <i>App, Aplp1</i> and <i>Aplp2</i> mRNAs were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in the denervated outer molecular layer (A) and in the granule cell layer (B). Only <i>App</i> mRNA was found to be upregulated in the granule cell layer. Similarly, mRNA expression levels of <i>Adam10</i>, <i>Adam17</i> and <i>Bace1</i> were determined by RT-qPCR in the denervated outer molecular layer (C) and in the granule cell layer (D). Significant upregulation of <i>Adam10</i> and <i>Adam17</i> mRNAs were seen in the denervated outer molecular layer (C). In addition, <i>Adam10</i> mRNA was found to be increased in the granule cell layer (D). (n = 4–6 animals each; t-test (two-tailed), * = p≤0.05).</p

Entorhino-hippocampal denervation model.

<p>(A) Schematic of a horizontal brain section illustrating the entorhino-hippocampal denervation model. The perforant path (green) originates from stellate neurons in the entorhinal cortex (EC) and terminates on distal dendritic segments of granule cells (gray) in the outer molecular layer (oml) of the dentate gyrus (DG). The transection site of the perforant path is indicated by a dashed line. (B, C) Layer-specific denervation of the dentate oml following unilateral transection of the perforant path. Fluoro-Jade C staining of the hippocampus contralateral (B) and ipsilateral (C) to the lesion side reveals degenerating axons in denervated areas of the dentate molecular layer at 7 days post lesion. Arrowheads point to the border between the denervated oml and non-denervated inner molecular layer (iml). Nuclei were counterstained with Hoechst 33342. gcl: granule cell layer; h: hilar region. Scale bar: 200 µm.</p

Reactive astrocytes upregulate ADAM10 in the dentate gyrus 7 days after entorhinal denervation.

<p>ADAM10 immunofluorescence of the hippocampus contralateral (A, C) and ipsilateral (B, D) to the lesion side revealed an upregulation of ADAM10 in the denervated outer molecular layer (oml) of the dentate gyrus. Arrowheads (in B) point to the border between the denervated oml and the non-denervated inner molecular layer (iml). Confocal analysis of sections double-stained for ADAM10 (E, F; red), the astrocytic marker GFAP (E; in yellow/green) and the microglia marker IBA1 (F; green) revealed that increased ADAM10 immunofluorescence (D–F, arrows) is associated with reactive astrocytes (E, arrows), but not with activated microglia (F, stars) following entorhinal denervation. Nuclei were counterstained with DRAQ5. h: hilar region. Scale bars: (A) 200 µm; (C) 25 µm.</p

Microarray analysis of candidate genes in the dentate outer molecular layer and granule cell layer after entorhinal denervation.

<p>Microarray analysis of candidate genes in the dentate outer molecular layer and granule cell layer after entorhinal denervation.</p

TaqMan assays used in the present study.

<p>TaqMan assays used in the present study.</p

Deregulated miRNAs involved in the immune response in ALK+ALCL.

<p>MiRNAs with known functions in the immune response [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117780#pone.0117780.ref062" target="_blank">62</a>] and deregulated in ALK+ ALCLs in our study are shown (red: downregulated miRNAs in ALK+ ALCL, green: upregulated miRNAs in ALK+ ALCL). The miR-17~92 cluster comprises the 6 miRNAs 17, 18a, 19a, 20a, 19b and 92a. The table shows the deep sequencing results of these miRNAs as base mean values from triplicates. CLP, common lymphocyte progenitor; CMP, common myeloid progenitor; DC, dentritic cell; DP, double positive T cell; DN, double negative T cell; GMP, granulocyte monocytic progenitor; HSC, hematopoietic stem cell; MDP, myeloid dendritic progenitor.</p

Comparative relative miRNA expression of miRNAs miR-146b-5p, miR-203, miR-181a* and miR-181a after C/EBPβ down-regulation—deep sequencing and RT-qPCR of ALK+ ALCL cell lines.

<p>Quantification of three by C/EBPβ knockdown significantly regulated miRNAs miR-146b-5p (upper panel), miR-203 (second panel), miR-181a* (third panel) and additionally miR-181a (lower panel), in ALK+ ALCL cell lines. (<b>A</b>) Deep sequencing results of ALK+ ALCL cell lines transduced with pF or pF-C/EBPβ shRNA. Results are depicted as base mean values from triplicates. (<b>B</b>) RT-qPCR analysis of miRNAs 146b-5p, 203, 181a* and 181a in pF and pF-C/EBPβ (shaded) transduced ALK+ ALCL cells four days after infection. Values were normalized to RNU6B and data were analysed according to the 2^-ΔΔCp method. Results are depicted as miRNA levels relative to untreated SUDHL-1 cells. Error bars indicate standard deviation of triplicates.</p

Analysis of C/EBPβ regulated miRNAs.

<p>(<b>A</b>) Western Blot analysis of the C/EBPβ isoforms LAP* and LAP in the transduced SR786 cells three days after infection. Each lane of the Western Blot contains 20 μg protein extract. Tubulin was used as loading control. SR786 = uninfected cells, pRRL = empty virus, pRRL-LAP* = virus containing the C/EBPβ isoform LAP* sequence, pRRL-LAP = virus containing the C/EBPβ isoform LAP sequence. (<b>B</b>) RT-qPCR analysis of miRNAs 146b-5p, 203, 181a* and 181a in untreated, mock-treated and pRRL.PPT.SF.i2GFPp (containing LAP and LAP* isoforms) transduced ALK+ ALCL cell line SR-786. Values were normalized to miR-106b and data were analysed according to the 2^-ΔΔCp method. Results are depicted as miRNA levels relative to untreated SR-786 cells. (<b>C</b>) RT-qPCR analysis of miRNAs 146b-5p, 203, 181a* and 181a in primary ALCL cases (4 ALK+ and 5 ALK- ALCL cases). For RT-qPCR quantification values were normalized to miR-106b and data were analysed according to the 2^-ΔΔCp method. Results are depicted as miRNA levels relative to mean value of ALK+ ALCL levels. For statistical analysis of RT-qPCR results a Wilcoxon rank-sum test was used (*p<0.05).</p

Principal Component Analysis.

<p>2D scatter plot shows principal component analysis (PCA) of miRNA deep sequencing data. The two axes represent the first two principal components (PCs) from the principal component analysis. The values in brackets indicate the amount of variation in the data that can be explained by the PC. The percent of variation given by a particular PC is indicated in the axis label. Points are colored by sample type. Samples were analyzed in triplicates. The graph shows a clear separation by principal component 1 between normal T cells (grey) and ALCL cells. The principal component 2 separates ALK+ and ALK- ALCL (light blue).</p

Differentially expressed miRNAs in ALK+, ALK- ALCL and normal T cells.

<p>(<b>A</b>) The bar graph illustrates the numbers of significantly differentially expressed miRNAs between the combined three ALK+ ALCL cell lines SUDHL-1, KiJK and Karpas 299, the ALK- ALCL cell line Mac-1 and normal T cells. The 13 most significantly differentially expressed miRNAs between the investigated ALK- ALCL cell line and the three ALK+ ALCL cell lines (upper right table) and the 13 miRNAs most significantly differentially expressed between the investigated three ALK+ ALCL cell lines and normal T cells (left table) are additionally designated. Data in both tables are depicted as base means of triplicates and fold change and Benjamini-Hochberg corrected p-values (padj) are indicated. Lower right table lists the top 13 miRNAs, which are significant differentially expressed in ALK+ ALCL, ALK- ALCL and normal T cells and present highest differences in expression levels. (<b>B</b>) The Venn diagram represents significant differentially expressed miRNAs in ALK+ and ALK- ALCL in the three ALK+ ALCL cell lines SUDHL-1, KiJK and Karpas 299. The analysis reveals a common profile of 106 deregulated miRNAs. (<b>C</b>) The Venn diagram illustrates significant differentially expressed miRNAs in ALK+ ALCL and T cells in the three ALK+ ALCL cell lines SUDHL-1, KiJK and Karpas 299. The analysis shows a common profile of 228 deregulated miRNAs.</p