PNAS2020.Fig.2

Experimental design and small RNA sequencing. (A) Schematic showing the full study design. Step 1: Three rodent models of epilepsy were generated: IAKA (intraamygdala kainic acid-induced status epilepticus in C57BL/6 mice), PILO (pilocarpine-induced status epilepticus in NMRI mice), and PPS (perforant pathway stimulation-induced hippocampal lesioning in Sprague-Dawley rats). Step 2: Hippocampi were extracted at six (mice) or seven (rat) time points and processed for Ago2 immunoprecipitation and ...

PNAS2020.Fig.6

Combi-antimiR suppresses seizures in established epilepsy. (A) Relative levels of miRNAs measured 24 h after injection of combi-antimiR, confirming effective inhibition of all three targeted miRNAs (closed circles, male mice; open circles, female mice, pooled for statistics; all t test *P < 0.05, **P < 0.01; n = 5 mice [scramble] and 6 mice [combi-antimiR]). (B) Representative EEGs during SE induced by IAKA in mice pretreated 24 h previously with scramble or combi-antimiR (#, EEG artifacts [excl...

PNAS2020.Fig.7

Target identification and pathway enrichment analysis identified TGF-β signaling as a potential convergent mechanism of the seizure-modifying miRNAs. (A) Number of mRNAs targeted by each miRNA. One mRNA (thyroid hormone receptor beta) is targeted by all 5 miRNAs. A total of 59 mRNAs are targeted by the 3 seizure-modifying miRNAs, 19 of which are not targeted by either miR-27a-3p or miR-431 (SI Appendix, Table S3). All targets are listed in Dataset S2. (B) Significantly enriched Reactome pathways...

SciRep2017.fig5

(a) Each transcript identified by Upf1 RIP-seq is marked by a black dot on the scatter plot. Transcripts that were upregulated after SE and were validated using PCR are marked in red. (b-g) The distribution of reads along each transcript in control and SE samples from synaptoneurosomes is presented. Note the markedly higher levels of reads found on the indicated transcripts after SE. (h i-vii) qPCR analysis of RNA isolated from ipsilateral hippocampus from control and SE mice validating RIP-seq ...

NAR2019.Fig4

Gene biotype and abundance are correlated to fraction of expressed cells. In general, the fraction of cells in which a gene is expressed is related to the mean expression level of that gene; exceptionally, some low abundant genes are present in a large fraction of cells. RNA biotypes that are known to be more cell-type specifically expressed, such as lincRNAs, are expressed in fewer cel

FrontiersMG2021.fig4

Transcriptome characterization at DOFS. (A) Principal component analysis (PCA) of the different experimental groups based on the transcriptome changes of the 1000 most highly expressed circRNAs. (B) Number of differentially expressed circRNAs (upper panel) and mRNAs (lower panel) at the six experimental time points (p < 0.05) (DE, differentially expressed). (C) Scatter plot showing the comparison of circRNA and mRNA expression. The x-axis shows the number of reads mapped across the back-splice j...

FrontiersMG2021.fig5

Validation of the expression of selected circRNAs and their linear counterparts at the DOFS. (A) Heatmap showing relative expression changes of the most deregulated rat-specific circRNAs. (B) Characterization of selected circRNAs (circ_Arhgap4, circ_Nav3, circ_Spidr and circ_Neto1) showing exonic composition, length and exon number. Each blue box represents one exon and the different sizes of the different exonic lengths. (C) Schematic workflow of the pipeline used for the annotation of selected...

FrontiersMG2021.fig6

Bioinformatics analysis of circRNA/miRNA networks. (A) Predicted miRNA targets of circ_Arhgap4 and circ_Nav3 (miRDB predicted score > 60). Dotted lines represent high confidence (miRDB predicted score = 80) and minimum confidence (miRDB predicted score = 60) thresholds used in further selection. (B) Diagram showing predicted mRNA targets derived from the circRNA/miRNA (miRDB analysis, predicted score > 80). circRNAs are represented by the pink rectangles; predicted bound miRNAs by the yellow ova...

FrontiersMG2021.fig3

Differentially expressed circRNAs. Heatmap representation of circRNA detection, conservation and expression in the different experimental groups [1 day, 3 days, and 10 days after performant pathway stimulation (PPS); day of first spontaneous seizure (DOFS) and 30 days after DOFS; control]. All circRNAs with FDR < 0.05 for any time point relative to control group are shown. Expression (z-score) of each circRNA is represented in each cell, where z-score represents the number of standard deviations...

PNAS2020.Fig.1

Experimental design and small RNA sequencing. (A) Schematic showing the full study design. Step 1: Three rodent models of epilepsy were generated: IAKA (intraamygdala kainic acid-induced status epilepticus in C57BL/6 mice), PILO (pilocarpine-induced status epilepticus in NMRI mice), and PPS (perforant pathway stimulation-induced hippocampal lesioning in Sprague-Dawley rats). Step 2: Hippocampi were extracted at six (mice) or seven (rat) time points and processed for Ago2 immunoprecipitation and ...

FrontiersMG2021.fig2

Characterization of circRNA profiling in the PPS rat model. (A) Distribution of detected circRNAs in the rat perforant pathway stimulation (PPS) model in different circRNA categories (exonic, intergenic, single-exon and multi-exon). (B) Size distribution of different exonic circRNAs, n = 771. (C) Distribution of the number of circRNA isoforms per host gene. (D) Distribution of circRNAs across the different rat chromosomes. (E) Expression levels of detected circRNAs across the different experimen...

biomedicines,22020.fig006

Characterization of WT SC-derived EV cargo in terms of miRNA content. Upset plot that shows the instances where more than 2 miRNAs target more than 5 genes. The plot is made using the R package “UpSetR”. The blue bars on the left side of the plot show the number of targets predicted for each miRNA. The right-side bar plot show the number of genes targeted by only a single miRNA (single black dot in the lower right panel) or multiple miRNAs (multiple linked dots in the lower right panel).

biomedicines.22020.fig004

WT Schwann cell EV Pathway and Process analysis. (a) Pathway analysis for all genes predicted to be targets of 3 or more miRNAs. Hits from KEGG pathways with p-value < 0.05. Cancer hits were removed. (b) Pathway analysis for all genes predicted to be targets of 3 or more miRNAs. The top 20 hits from Gene Ontology Biological Process. (c) Pathway analysis for predicted targets of each individual miRNA. The 30 most significant hits from KEGG pathways are shown. The heatmap shows the level of signif...

biomedicines2020.fig006

Differential expression analysis of tsRNAs in MSCs and MSC-EVs. (A) Heat map shows the expression profile of tsRNAs with unsupervised clustering of sample origin (B) Differential expression analysis of tsRNAs using DESeq2. The miRNAs with Abs (log2FoldChange) ≥ 1 and FDR < 0.05 are deemed as significantly changed and marked in red in the volcano plot.

biomedicines2020.fig004

Functional enrichment analysis for target genes of differentially expressed miRNAs in osteoblastogenesis. (A) miRNAs identified in both ASCs and BMSCs. (B) miRNAs identified in both BMSCs and BMSC-EVs. (C) miRNAs identified only in BMSC-EVs. The Y-axis shows the annotation categories (e.g., KEGG pathways) and the X-axis shows the miRNAs. The color of the dots represents the adjusted p values (FDR) and the size of the dots represents gene ratio (the number of miRNA targets enriched in each catego...

biomedicines.22020.fig010

Transgenic Schwann cell EV cargo characterization (lacking p75NTR or sortilin expression). (a) UpSet plot showing the number of predicted target genes found to be targeted by individual miRNAs or by multiple miRNAs. The plot is made using the R package “UpSetR”. The blue bars on the left side of the plot show the number of targets predicted for each miRNA. The right-side bar plot shows the number of genes targeted by only a single miRNA (single black dot in the lower right panel) or multiple miR...

biomedicines2020.fig003

Differential expression analysis of miRNAs in MSCs and MSC-EVs in osteoblastic differentiation. (A) Principal component analysis (PCA) analysis (filled circle, cells; triangles, EVs; red, ASC; green, BMSC; numbers, different cell donors); (B) Differential expression analysis of miRNAs using DESeq2. The miRNAs with Abs (log2FoldChange) ≥ 2 and p value < 0.05 are deemed as significantly changed and marked in red in the volcano plot.

biomedicines.22020.fig009

Differentially expressed miRNAs in EVs derived from Ngfr−/− and Sort1−/− compared with EVs from WT Schwann cells (SCs). (a) The 45 most significantly differentially expressed miRNAs (not multiple-testing corrected) are shown as a heatmap covering all samples from WT, Ngfr−/− and Sort1−/− SCs. Top annotation shows Sort1−/− in orange, Ngfr−/− in yellow and control WT as blue. Shown are z scores of log2-transformed RPM values. (b) Volcano plot for EV miRNAs from Ngfr−/− versus WT comparison and (c)...

SciRep2017.fig4

Upf1-bound transcripts whose log2-fold expression was decreased by 1.5 fold or was completely absent from SE samples and whose log2-fold expression was increased 1.5 fold or was completely absent from control samples were used to investigate over- and under-represented GO terms. Numbers of genes in each group are displayed in inset Venn diagram. (a) Biosynthetic and mRNA processes were over-represented in those genes only expressed or decreased in control SN sample. (b) Terms relating to biosynt...

SciRep2017.fig3

(a) Schematic of RIP-seq experimental design. Original drawing by author Claire Mooney. (b) Immunoblot showing Upf1 pull-down in a separate experiment in samples from synaptoneurosome (SN) and cytoplasm compartments generated from hippocampi from control and SE (8 h) mice. Representative blots have been cropped to reduce unnecessary area. (c) Raw reads in each sample. (d) Distribution of raw reads from control and SE samples of cytoplasm and synpatoneurosome fractions mapped to exonic gene regio...

PNAS2020.Fig.3

Identification of common-to-all model miRNAs. (A) Graphs show the overlap of up-and down-regulated miRNAs between the three models at various phases of epilepsy development. (B) The miRNAs with consistent up-regulation in all three models, common-to-all miRNAs, are further highlighted. (C) Examples of the expression data from the PPS model for the common-to-all miRNAs up-regulated in chronic epilepsy (excluding miR-146a-5p and miR-132-3p)