Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Characterization of EVs Isolated from Fetal Mouse Neurosphere Cultures 2A) Transmission electron micrograph images of CD63-immunogold-labelled EVs. Red circles indicate immunogold puncta, indicating CD63-like immunoreactivity. 2B) Frequency Distribution of EV diameter. Inset depicts Nanosight image of EVs derived from NSCs. 2C) Quantitative immunoblot analysis of relative protein levels of positive EV markers Tsg101, AnnexinVI, Rab5, CD63, CD9. 2D) negative EV markers Calnexin, Cytochrome C, Ago2 from NSCs and isolated EVs from the culture media of these NSCs. n = 3 to 7 samples per group. Significance was determined using unpaired t-test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, # p < 0.08. 2E) Representative immunoblots of positive EV markers, Tsg101 and annexin VI (green fluorescence). 2F) Representative immunoblots of negative EV markers, Ago2 and Calnexin (green fluorescence). Red bars at the base of each immunoblot denote cell lysates (CL) and green bars denote EV-derived protein lysates (EV).

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Isolation, Transmission Assay, Derivative Assay, Western Blot, Fluorescence

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: WGCNA-based Comparisons of the Protein Content of EVs Relative to Parental NSCs. WGCNA of proteins expressed in all 36 samples, shows that EVs and their parent cells have distinct protein networks and correlations with the trait of “location” (Cell vs. EV) contributing to the majority of the difference between clusters. Other traits including “pregnancy” (cells derived from three separate pregnancies), fetal sex (female or male), and “alcohol” (ethanol exposure at 0, 120 or 320 mg/dL) were smaller contributors to the overall composition of identified networks. 3A) Topological overlap matrix (TOM) plot for visualizing the weighted gene (protein) co-expression network, where the topological overlap considers each pair of proteins' similarity in relation to all other proteins in the network. Modules are defined by hierarchical clustering, visualized by dendrograms and module color notation. Each row or column corresponds to a single protein, where lighter red denotes low topological overlap and darker red denotes high topological overlap. 3B) Multidimensional scaling (MDS) plot showing high gene overlap and similarity across brown, yellow, blue, and green modules, while the turquoise module, containing a majority of proteins, did not exhibit overlap or similarity to other modules. 3C) Heatmap of WGCNA module significance (MS) correlations with sample traits. In the rows, modules are shown named by their corresponding colors and across the columns are the sample traits of interest. Numbers in the table correspond to the correlation coefficients between the module eigengene (ME) and the specific trait, with p-value in parentheses. The degree of correlation is illustrated with the color legend; the more intense the box color, the more positively (red) or negatively (blue) correlated is the module with the trait. The results of this analysis show that location (cell vs. EV) is the most important determinant of module identity, with the turquoise module identifying cells, and the yellow, brown, blue and green modules identifying EVs.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Derivative Assay, Expressing

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Lysosome Subcellular Located Proteins in EVs and Cells 7A) Volcano plot depicting relative expression of lysosome-localized proteins (based on UniProt Knowledgebase), with the expression of individual proteins z-score transformed across samples. 7B) Graph showing changes in average protein content as represented by the average z-score calculated for each sex and sample type (EV or cell). Pairwise analysis showed that, contrary to predictions, female fetal NSC-derived EV's expressed equivalent levels of lysosome-localized proteins compared to parental NSCs, whereas male NSC-derived EVs contained a smaller quantity of lysosome-localized proteins compared to parental NSCs. ∗∗∗ p < 0.001; ns, not significant; n = 9 for each group. 7C) Top EV enriched lysosome-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples. 7D) Top cell enriched lysosome-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Expressing, Transformation Assay, Derivative Assay

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Endosome Subcellular Located Proteins in EVs and Cells 8A) Volcano plot depicting relative expression of endosome-localized proteins (based on UniProt Knowledgebase), with the expression of individual proteins z-score transformed across samples. 8B) Graph showing changes in average protein content as represented by the average z-score calculated for each sex and sample type (EV or cell). Pairwise analysis showed that, contrary to predictions, male fetal NSC-derived EV's expressed equivalent levels of endosome-localized proteins compared to parental NSCs, whereas female NSC-derived EVs exhibited a trend (#, p = 0.06) towards a larger quantity of endosome-localized proteins compared to parental NSCs; n = 9 for each group. 8C) Top EV enriched endosome-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples. 8D) Top cell enriched endosome-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Expressing, Transformation Assay, Derivative Assay

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Membrane Subcellular Located Proteins in EVs and Cells 9A) Volcano plot depicting relative expression of membrane-localized proteins (based on UniProt Knowledgebase), with the expression of individual proteins z-score transformed across samples. 9B) Graph showing changes in average protein content as represented by the average z-score calculated for each sex and sample type (EV or cell). Pairwise analysis showed that, contrary to predictions, male fetal NSC-derived EV's expressed equivalent levels of membrane-localized proteins compared to parental NSCs, whereas female NSC-derived EVs exhibited a significantly larger (∗, p < 0.056) quantity of membrane-localized proteins compared to parental NSCs; n = 9 for each group; ns, not significant. 9C) Top EV enriched membrane-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples. 9D) Top cell enriched membrane-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Membrane, Expressing, Transformation Assay, Derivative Assay

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Ethanol Treatment Increases the Complexity of Correlated Protein Networks in EVs. 11A) Topological overlap matrix plots depict weighted gene/protein co-expression networks in EVs (upper panel) or parental cell samples (lower panel), separated by ethanol treatment condition, 0 mg/dL, left panels; 120 mg/dL, center panels; 320 mg/dL, right panels. Modules were defined by hierarchical clustering as depicted by dendrograms and designated by a unique color identity. 11B) Bar graph depicts number of identified modules for EV and cell samples, as a function of ethanol exposure dose. Chi-squared tests determined that there was an ethanol dose-related increase in the number of modules in EVs relative to parental NSCs. ∗∗∗∗, p < 0.0001; #, p = 0.072, trending towards significance.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Expressing

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Effect of exposing NSCs to a Dose Range of Ethanol on the Proteome of Secreted EVs and Cells Scatter plots of effect size (Hedges' g) vs. normalized protein expression level, document that statistical significance ( p < 0.05) is independent of protein expression, but generally associated with increased effect size. 12A,B) Relationship between effect size and statistical significance in EVs from 120 mg/dL-treated NSCs (12A) or 320 mg/dL-treated NSCs (12B) relative to control (0 mg/dL) NSCs. These analyses show that more differentially regulated EV proteins also exhibited a positive effect size, indicative of upregulation in EVs. 12C,D) Relationship between effect size and statistical significance in parental NSCs treated with 120 mg/dL (12C) or 320 mg/dL (12D) relative to control (0 mg/dL) NSCs. In contrast to EVs, these analyses show that more differentially regulated parental NSC proteins also exhibited a negative effect size, indicative of down-regulation. Horizontal blue-dotted line represents the average protein expression level in EV (12A,B) or parental cell (12C,D) samples. Vertical purple-dotted lines denote effect size values of -0.8–0.4, -0.2, +0.2, +0.4, +0.8. Positive effect size signifies increased expression of a protein, and negative effect size signifies decreased expression of a protein, in treatment group vs. control group. Each data point represents a protein, with red color for significant p-value; n = 6 samples per group; paired t -test, p < 0.05. 12E,F) Volcano plots of the relationship between effect size due to treatment and p-value. Each data point represents protein enrichment in EVs relative to parental cells (EV/Cell) between moderate ethanol-treated (120 mg/dL, 12E) or heavy ethanol-treated (320 mg/dL, 12F) groups compared to the control group (0 mg/dL). These data show that proteins that were significantly enriched in EVs due to ethanol exposure were also depleted in parental NSCs. Blue triangles above the horizontal dotted line represent proteins for which the effect size had a non-zero containing 95% confidence estimate that were also significantly affected by ethanol exposure by paired t-test. Green points below the horizontal dotted line represent proteins for which the effect size had a non-zero containing 95% confidence estimate but were not significantly affected by ethanol exposure by paired t-test. Red points above the horizontal dotted line represents proteins for which the effect size had a zero containing 95% confidence estimate but were significantly affected by ethanol exposure by paired t-test; n = 6 samples per group; paired t -test, p < 0.05.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Expressing, Control, Protein Enrichment

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: ScRNAseq of GD 14.5 Murine Developing Cortex Shows that mRNAs for Ethanol-sensitive EV-Enriched Proteins are Abundant in Ventricular Zone Cell Lineages. 14A) tSNE plot of clusters identified as part of VZ, SVZ, or TPC lineages. Data extracted from NCBI/GEO (GSE158747) ( 12 ). 14B) tSNE plot classifying VZ, SVZ, TPC lineages. 14C,D) Composite mRNA transcript expression for proteins significantly enriched in EVs, with Hedges' g > +0.4 for EVs obtained from 120 mg/dL (14C) and 320 mg/dL (14D)-treated NSCs. 14E,F) The application of a threshold cutoff of >log 2 4 for composite transcript expression shows that in vivo , neural progenitor cells of the VZ are the principal contributors of proteins that are significantly enriched in EVs following exposure of parental NSCs to 120 mg/dL (14E) or 320 mg/dL (14F) of ethanol.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Expressing, In Vivo

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Fluorescent-Labeled EVs from Donor NSCs are Sequestered by Recipient NSCs 15A) Confocal photomicrograph showing the presence of MemBrite-labeled EVs (red fluorescence) purified from donor NSCs within the cytoplasm of naïve NSCs counterstained with DAPI (blue) to visualize nuclei. 15B) Flow cytometric analysis shows that compared to unstained cells, MemBrite readily stains NSCs. Purified EVs stained with MemBrite are also readily taken up by naïve recipient cells, whereas no stain was observed in cells exposed to purified culture medium with MemBrite dye that underwent the same labeling and filtration process as isolated EVs; n = 6 to 13 samples per group; Kruskal-Wallis test for nonparametric one-way ANOVA, p < 0.0001; Dunn's multiple comparisons post-hoc test, ∗ p < 0.05.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Labeling, Fluorescence, Purification, Staining, Filtration, Isolation

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Functional Analysis of the Effects of Purified Control EVs, and EVs Purified from Ethanol-treated NSCs on the Behavior of Naïve Recipient NSCs. Following EV administration, oxidative metabolism was measured by the alamarBlue assay (16A), glycolysis was measured by the Lactate-Glo assay (16B), and apoptosis was measured by the activation of Caspase3/7 (16C) in naïve recipient NSCs; n = 5 to 23 samples per group; repeated-measures two-way ANOVA with Geisser-Greenhouse correction; Tukey's or Dunnett's multiple comparisons post-hoc test, ∗ p < 0.05, # p < 0.08. 16D,E,F) Quantification in donor, ethanol-treated and control NSCs, of oxidative metabolic activity (16D), glycolysis activity (16E), and Caspase 3/7 apoptosis activity (16F) following moderate (120 mg/dL) or high (320 mg/dL) ethanol exposure in NSCs; n = 5 to 23 samples per group; repeated-measures two-way ANOVA with Geisser-Greenhouse correction; Tukey's multiple comparisons post-hoc test, ∗ p < 0.05. 16G) Flow cytometry analysis of the proportion of cells in S-phase of the cell cycle, following addition of EVs derived from ethanol-treated or control NSCs, to naïve recipient NSCs; n = 19 samples per group; repeated-measures two-way ANOVA with Geisser-Greenhouse correction; Dunnett's multiple comparisons post-hoc test, ∗ p < 0.05, # p < 0.08. 16H) Representative flow cytometry image.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Functional Assay, Purification, Control, Alamar Blue Assay, Glo Assay, Activation Assay, Activity Assay, Flow Cytometry, Derivative Assay

Journal: eLife

Article Title: Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations

doi: 10.7554/eLife.74338

Figure Lengend Snippet: ( A ) Heatmaps showing the correlation between CUT&RUN profiles of H3K27ac and H3K27me3, and RNA-Seq in Hct116 WT cells. ( B ) Genome browser view of H3K27ac and H3K27me3 signal from Hct116 WT, Fbw7 R/+ , and Fbw7 −/− cells at a representative gene. ( C ) Peaks with increased (red) or decreased (blue) H3K27ac signal in Hct116 Fbw7 −/− and Fbw7 R/+ cells compared to WT cells. Differential sites indicated as a percent of total H3K27ac peaks in Hct116 WT cells. ( D ) Percentage of H3K27ac peaks located within different gene regions. ( E ) Sequence logo for AP-1 motif enriched in H3K27ac peaks increased in Fbw7 −/− cells (E value=1.6e−3). See , , and . has the complete MEME output and details on the FIMO analysis. CUT&RUN, cleavage under target and release using nuclease; WT, wild-type. Figure 2—source data 1. H3K27ac differential sites. This Excel file includes lists of peaks with increased and decreased H3K27ac signal in Hct116 Fbw7 −/− and Fbw7 R/+ relative to WT. Fold change and FDR listed for each peak. WT, wild-type. Figure 2—source data 2. Summary of CUT&RUN differential sites. This Excel file includes a summary (total number of differential sites, percentage, and number of annotated genes) of H3K27ac, Jun, and Myc differential sites in Hct116 cells and Jun differential sites in U5-NSCs. CUT&RUN, cleavage under target and release using nuclease; NSC, neural stem cell.

Article Snippet: Cell line ( H. sapiens ) , U5 human neural stem cells (NSCs) , Jackson Laboratory , B6.129P2Gpr37tm1Dgen/J , Primary cells.

Techniques: RNA Sequencing, Sequencing

Journal: eLife

Article Title: Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations

doi: 10.7554/eLife.74338

Figure Lengend Snippet: ( A ) Western blot showing Fbw7; samples 1–4: U5 NSCs with sgRNA targeting Fbw7 exons 3, 4, and 9, and all three exons in one pool; samples 5–6: U5 NSCs with control sgRNA 1× and 3×; and sample 7: Hct116 WT. ( B ) Sequence logo of AP-1 motif enriched in Jun peaks in U5 NSCs (E value=1.2e−146). CUT&RUN, cleavage under target and release using nuclease; NSC, neural stem cell; WT, wild-type.

Article Snippet: Cell line ( H. sapiens ) , U5 human neural stem cells (NSCs) , Jackson Laboratory , B6.129P2Gpr37tm1Dgen/J , Primary cells.

Techniques: Western Blot, Control, Sequencing

Journal: eLife

Article Title: Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations

doi: 10.7554/eLife.74338

Figure Lengend Snippet: ( A ) Cell proliferation was monitored with an IncuCyte for 81 hr. Data are shown as mean ± SEM. Proliferation was measured at each time point normalized to percent confluence at time 0. Doubling time, WT 61.3 hr and Fbw7 −/− 88.82 hr. ( B ) Flow cytometry analysis of DNA replication in Fbw7 mutant cells. Density plots of EdU versus DNA content (DAPI) in NSCs. Single cells were identified by gating events on DAPI-H/DAPI-A. Cells were labeled with EdU prior to harvest and were later fixed and immuno-stained for flow cytometry. NSC, neural stem cell; WT, wild-type.

Article Snippet: Cell line ( H. sapiens ) , U5 human neural stem cells (NSCs) , Jackson Laboratory , B6.129P2Gpr37tm1Dgen/J , Primary cells.

Techniques: Flow Cytometry, Mutagenesis, Labeling, Staining

Journal: eLife

Article Title: Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations

doi: 10.7554/eLife.74338

Figure Lengend Snippet: ( A ) Clustering analysis separates differentially expressed protein-coding genes in NSCs into two groups. Heatmap shows the intensity of expression of each gene (y-axis) for three replicates per cell type (x-axis). Three replicates were from two independently engineered cell samples. ( B ) TFs, pathways, and GO terms enriched in upregulated genes in Fbw7 −/− NSCs. ( C ) Sites with increased (red) and decreased (blue) Jun in Fbw7 −/− NSCs compared to WT. ( D ) Nondifferential and differential Jun peaks located within each gene feature. ( E ) Fold change of CIITA and MHC Class II genes in Fbw7 −/− NSCs compared to WT. FDR values are given at the top of each bar. n=3. ( F ) Quantitative RT-PCR analysis of MHC Class II (HLA-DRA, HLA-DRB, HLA-DPA, and HLA-DPB) expression in Fbw7 −/− NSCs. Mean fold change in Fbw7 −/− cells with respect to WT cells. Error bars=SEM, n=3. ( G ) Genome browser view of Myc, Jun, and H3K27ac occupancy on CIITA regulatory regions in WT and Fbw7 −/− NSCs. Black scale bar=8 kb. See – and . GO, gene ontology; TF, transcription factor; WT, wild-type. Figure 6—source data 1. Confirming loss of Fbw7 in U5-NSC Fbw7 −/− cells. This folder contains the original western blots for . Western blots that confirm the loss of Fbw7 in two others separately performed nucleofection reactions are also included. Figure 6—source data 2. Differential expression analysis of U5-NSC RNA-Seq. This Excel file contains the differential analysis output of U5-NSC RNA-Seq data from control (Ctrl23, Ctrl4.1, and Ctrl4.2) and Fbw7 −/− (Fb23, Fb4.1, and Fb4.2) cells. DE=0, not differentially expressed (DE) in the mutant compared to WT; DE=1, DE in the mutant compared to WT. NSC, neural stem cell; WT, wild-type. Figure 6—source data 3. Enrichr output for U5-NSC differentially expressed genes. This Excel file includes the TRANSFAC & JASPAR, MSigDB, and GO term outputs enriched in differential (upregulated or downregulated) genes in Fbw7 −/− NSCs. GO, gene ontology; NSC, neural stem cell. Figure 6—source data 4. Jun differential sites in U5-NSCs. (U5F=Fbw7 −/− and U5W=WT). NSC, neural stem cell; WT, wild-type. Figure 6—source data 5. Original gels for . Figure 6—source data 6. Quantitative RT-PCR analysis of MHC Class II genes in NSCs. NSC, neural stem cell. Figure 6—source data 7. Original western blots for .

Article Snippet: Cell line ( H. sapiens ) , U5 human neural stem cells (NSCs) , Jackson Laboratory , B6.129P2Gpr37tm1Dgen/J , Primary cells.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, RNA Sequencing, Control, Mutagenesis

Journal: eLife

Article Title: Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations

doi: 10.7554/eLife.74338

Figure Lengend Snippet: ( A ) Hierarchical clustering of DE protein-coding genes in Hct116. Expression level of the same genes in NSCs is also mapped. Genes that are not captured in NSC are in gray. Log 2 FC of DE genes specific to Hct116 cells was higher than the DE genes shared by both Hct116 and NSCs (Wilcoxon test: p=5.33e−16). ( B ) Hierarchical clustering of DE protein-coding genes in NSC. Expression level of the same genes in Hct116 cells is also mapped. Log 2 FC of DE genes specific to NSCs was higher than the DE genes shared by both Hct116 and NSCs (Wilcoxon test: p=3.727e−10). NSC, neural stem cell.

Article Snippet: Cell line ( H. sapiens ) , U5 human neural stem cells (NSCs) , Jackson Laboratory , B6.129P2Gpr37tm1Dgen/J , Primary cells.

Techniques: Expressing

Journal: eLife

Article Title: Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations

doi: 10.7554/eLife.74338

Figure Lengend Snippet: ( A ) Flow cytometry analysis of HLA-DR in Fbw7 mutant NSCs. Density plots of side scatter (SSC) versus PE HLA-DR. ( B ) Western blot detecting HLA-DR/DP/DQ in NSCs. Protein intensity of HLA band was measured using ImageJ in comparison to the loading control. HLA protein is ~3-fold higher in Fbw7 −/− cells in comparison to WT. NSC, neural stem cell; WT, wild-type.

Article Snippet: Cell line ( H. sapiens ) , U5 human neural stem cells (NSCs) , Jackson Laboratory , B6.129P2Gpr37tm1Dgen/J , Primary cells.

Techniques: Flow Cytometry, Mutagenesis, Western Blot, Comparison, Control

Journal: eLife

Article Title: Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations

doi: 10.7554/eLife.74338

Figure Lengend Snippet:

Article Snippet: Cell line ( H. sapiens ) , U5 human neural stem cells (NSCs) , Jackson Laboratory , B6.129P2Gpr37tm1Dgen/J , Primary cells.

Techniques: Reverse Transcription, SYBR Green Assay, Software, Imaging