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

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

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

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

Comparative relative miRNA expression of miRNAs miR-342-3p, miR-146a and miR-29c—deep sequencing, RT-qPCR of cell lines and primary samples.

<p>Quantification of three differentially expressed miRNAs miR-342-3p (upper panel), miR-146a (middle panel) and miR-29c (lower panel) between ALK+ ALCL, ALK- ALCL, T cells (isolated from 3 healthy donors), and normal lymph nodes, respectively. (<b>A</b>) Deep sequencing results. Data are depicted as base mean values from triplicates. (<b>B</b>) RT-qPCR validation analysis. Error bars indicate standard deviation of triplicates for cell lines and number of tested samples, respectively. (<b>C</b>) RT-qPCR validation analysis in primary ALCL cases and reactive lymph nodes (RLN). For RT-qPCR quantification values were normalized to miR-106b and data were analyzed according to the 2^-ΔΔCp method. Results are depicted as miRNA levels relative to mean value ALK+ ALCL levels. For statistical analysis of RT-qPCR results a Wilcoxon rank-sum test was used (*p<0.05, **p<0.01).</p

MiRNAs significantly regulated by C/EBPβ knockdown in ALK+ ALCL cell lines.

<p>MiRNA expression profiling of the three ALK+ ALCL cell lines SUDHL-1, KiJK and Karpas 299 with (pF-C/EBPβ) and without (pF) C/EBPβ down-regulation. (<b>A</b>) The circle diagram indicates the number of miRNAs regulated by C/EBPβ knockdown in the particular ALK+ ALCL cell line. (<b>B</b>) C/EBPβ-signature of SUDHL-1 cells derived by C/EBPβ-shRNA. Heatmap shows the expression pattern of the 53 miRNAs significantly regulated by C/EBPβ in SUDHL-1 cells, depicting transduced cells with C/EBPβ-shRNA and controls in triplicates. (<b>C</b>) The Venn diagrams illustrate the number of significantly down-regulated (left) and up-regulated (right) miRNAs in the ALK+ ALCL cell lines after transduction with C/EBPβ-shRNA. (<b>D</b>) The table shows from all miR-181 family members the miRNA expression levels (base mean of triplicates) of the three ALK+ ALCL cell lines SUDHL-1, KiJK and Karpas 299 with (pF-C/EBPβ) and without (pF) C/EBPβ knockdown as well as the ALK- ALCL cell line Mac-1 and normal T cells. (<b>E</b>) RT-qPCR analysis of miR-181a and miR-181c expression in ALK+ (SUDHL-1, KiJK and Karpas 299) and ALK- (Mac-1) ALCL cell lines and T cells. Error bars indicate standard deviation of triplicates for cell lines and number of tested samples, respectively. 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 T cell levels. For statistical analysis of RT-qPCR results a Wilcoxon rank-sum test was used (*p<0.05).</p

Numbers of reads and identified miRNAs obtained by deep sequencing.

<p>Numbers of unfiltered and filtered reads, percentage of filtered reads and numbers of miRNA reads with indication of percentage as well as numbers of different miRNAs identified are depicted for each sample applied to deep sequencing.</p><p>Numbers of reads and identified miRNAs obtained by deep sequencing.</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

Additional file 1: of ATRX immunostaining predicts IDH and H3F3A status in gliomas

Overview of ATRX staining results in tissue microarrays and full slides in the same tumors. (XLS 36 kb

Additional file 2: of ATRX immunostaining predicts IDH and H3F3A status in gliomas

Additional statistical data for IDH, MGMT, WHO, 1p/19q codeletion, age and diagnosis. (XLSX 11 kb