Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: Study Design Under BSL-4 containment, we collected blood samples from a total of 21 rhesus monkeys at multiple days post-EBOV inoculation, extracted peripheral blood mononuclear cells (PBMCs), and profiled single-cell transcriptomes and 42 protein markers using Seq-Well and CyTOF. Seq-Well quantifies both host (black) and viral (red) RNA expression, allowing comparisons between infected and bystander cells. Daily clinical parameters (body temperature, clinical signs, and body weight) were also collected for each animal, and complete blood counts were obtained for each blood draw. See also Figure S1 A and .

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: RNA Expression, Infection

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: Quantification of Cytokine Expression and Enrichment of Response Signatures, Related to and ( A ) Average expression values (log e TP10K) of literature-annotated cytokines (columns) across cell types and stages of acute EVD (rows). Values are plotted as a ratio relative to the maximum across cell types and stages. Values that are statistically different from baseline (p < 0.05) are indicated with a blue star. ( B ) Heatmap of rank-sum test statistics for comparison of differential expression log fold-changes of genes in a gene set (rows) compared to genes not in the set. The log fold-changes were defined from differential expression profiles of each cell type at each EVD stage (columns) relative to baseline. Five gene sets were tested — three from the Hallmark database (IFN ALPHA, IFN GAMMA, and TNF ALPHA VIA NFKB) ( Liberzon et al., 2015 ) and 2 constructed from the hallmark sets, as uniquely IFNα-regulated genes in “IFN ALPHA” but not “IFN GAMMA” (“IFN ALPHA - GAMMA”), and vice versa for uniquely IFNγ-regulated (“IFN GAMMA - ALPHA”). See also . ( C ) Fold change (log 2 scale) in average HLA-DR CyTOF intensity on B cells at each DPI relative to baseline for each PBMC sample. Colored lines connect serial samples from the same NHP.

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Expressing, Comparison, Quantitative Proteomics, Construct

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: ISG Suppression, Co-expression of CD14 and CD16, and Expression of Macrophage Genes Are Associated with Monocyte Infectivity (A) Differential expression between infected and bystander monocytes from DPI 5–8. Genes are colored by membership in sets of genes (Mac. Up/Down = up- or downregulated during in vitro differentiation of monocytes into macrophages). See also . (B) UMAP embedding of monocyte gene expression data, colored by (left-to-right) DPI, CD16 expression (log e TP10K), CD14 expression (log e TP10K), and percentage of cellular transcripts mapping to EBOV. (C) Smoothed expression (log e TP10K) of CD14 and CD16 for monocytes during EVD. Boxes: CD14 + , CD16 + , DN, and DP subsets described in the text; numbers: percentage of cells in each subset at that EVD stage. See also A and S5B. (D) CD14 and CD16 protein expression (CyTOF intensity) on monocytes at each DPI. Bivariate kernel density plot with 200 randomly sampled cells is overlaid as a scatterplot. See also Figure S5 C. (E) CD14 and CD16 protein expression (CyTOF intensity) on monocytes in a case of human EVD, colored by Ki67 protein expression for multiple days after symptom onset. See also Figure S5 D. (F) Percentage of assignment of NHP CD14/CD16 subsets at each EVD stage to human myeloid reference populations (BM-MP: bone marrow monocyte progenitors, PBMC-CD16 + : circulating CD16 + monocytes, PBMC-CD14 + : circulating CD14 + monocytes). See also E–S5K. (G) Percentage of infected monocytes in each CD14/CD16 subset in late EVD. Error bars: 95% CI on the mean based on 1,000 bootstraps. (H) Association between macrophage score (x axis) and percentage of infected cells (left y axis, red) and expression of the differentiation marker NR1H3 (right y axis, blue, log e TP10K). We ordered monocytes from late EVD by macrophage score, and averaged percentage of infected cells and NR1H3 expression within 400-cell sliding windows. See also A–S6C. (I) MX1 expression (log e TP10K) in monocytes at baseline, and uninfected bystanders or infected cells in late infection. Boxes: median and interquartile range; whiskers: 2.5 th and 97.5 th percentiles. Statistical significance was assessed by rank-sum test. See also Figure S6 D. (J) Scatterplot of ISG score (y axis) versus percentage of cellular transcripts mapping to EBOV (x axis) for infected monocytes in late EVD (DPI 6–8). Statistical significance was assessed by Spearman ρ.

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Expressing, Infection, Quantitative Proteomics, In Vitro, Gene Expression, Marker

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: Extended Characterization of Interferon and Double-Negative CD14 – CD16 – Monocytes, Related to Figure 5 ( A ) Clustermap of pairwise Pearson correlations between cell type clusters at baseline and late EVD. Correlations are computed on average log e TP10K expression values of overdispersed genes. DN and DP monocytes at late EVD are more similar to monocytes (including baseline CD14+s) than other cell types. ( B ) Scatterplot of MAGIC-smoothed expression values (log e TP10K) of CD14 and CD16 for monocytes in baseline, early, mid, and late disease stages. Cells are colored by smoothed expression levels of MKI67 (the gene coding for Ki67 protein). Boxes: CD14+, CD16+, DN, and DP subsets described in the text; numbers: percentage of cells falling into each subset. ( C ) Scatterplot of protein expression (CyTOF intensity) of CD14 and CD16 for 1,000 randomly sampled monocytes at each DPI. Cells are colored by Ki67 expression. Boxes: CD14+, CD16+, DN, and DP subsets described in the text; numbers: percentage of cells falling into each subset. ( D ) Scatterplot of protein expression (CyTOF intensity) of CD14 and CD16 for monocytes during human EVD. Left: monocytes from healthy human controls. Right: monocytes from 3 EVD cases (S1, S2, and S3) at various days post symptom onset. Cells are colored by Ki67 marker intensity. Boxes: CD14+, CD16+, DN, and DP subsets described in the text; numbers: percentage of cells falling into each subset. ( E ) UMAP embedding of healthy human PBMCs dataset, colored by annotated cluster assignment, based on known marker genes. (Plasma.: Plasmablast). ( F ) UMAP embedding of healthy bone marrow cells, colored by cluster assignment, based on marker genes. (HSC: hematopoietic stem cell, Plasma.: Plasmablast, Megakar.: Megakaryocyte, Mono/DC: monocyte and dendritic cell, BM-Macro: bone marrow macrophage). ( G ) UMAP embedding of sub-clustered HSC and monocyte/dendritic lineage cells. (BM: bone marrow, MP: monocyte progenitor) ( H ) Same UMAP embedding as Figure S5 G, but colored by the cluster identity of their nearest neighbor in the human PBMC dataset ( Figure S5 E). ( I ) UMAP embedding of the merged reference dataset of healthy bone marrow HSCs and monocyte lineage cells and PBMCs. Left sub-panel is colored by cluster assignment. Right sub-panels are colored by marker gene expression (log e TP10K). ( J ) Expression profiles of selected genes for human bone marrow monocyte progenitors (BM-MPs) and human circulating monocytes (PBMC-Monos). Circle area: percentage of cells in which the gene was detected; color: average expression ( Z -normalized log e TP10K). ( K ) Expression profiles of selected genes for NHP monocyte subsets at baseline or late EVD for orthologs of the genes in (J). Circle area: percentage of cells in which the gene was detected; color: average expression level ( Z -normalized log e TP10K). CD34 is grayed out because it is detected in <10 cells.

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Expressing, Marker, Clinical Proteomics, Gene Expression

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: Viral Transcriptional Dynamics of Infected Monocytes In Vivo and Ex Vivo (A) Schematic of EBOV challenge of PBMCs ex vivo . See also Figure S7 . (B and C) Percentage of cellular transcripts derived from EBOV (intracellular viral load) in monocytes from PBMCs inoculated with live virus ex vivo (B) or from PBMCs of NHPs infected in vivo (C). See also A–S8D. (D) Schematic of EBOV transcription. The viral RNA-directed RNA-polymerase transcribes each gene sequentially but occasionally releases the genomic RNA template, ending transcription. As a result, transcription frequency decreases from NP to L . (E and F) Proportion of each EBOV gene versus viral load (log 10 scale), ex vivo (E) or in vivo (F). We ordered infected monocytes by viral load and averaged the percentage of each viral gene over 50-cell sliding windows. Bands: mean ± 1 SD. See also E and S8F.

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Infection, In Vivo, Ex Vivo, Derivative Assay, Virus

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: EBOV Infection Downregulates Host Antiviral Genes and Upregulates Putative Pro-viral Genes (A and B) Association between host gene expression and viral load within infected monocytes from PBMCs 24 HPI treated with live virus ex vivo (A) or from PBMCs of NHPs in vivo on DPI 5–8 (B). See also . (C and D) Select negatively (C) and positively (D) associated genes in monocytes from ex vivo infections. We ordered infected cells by viral load and averaged gene expression (log e TP10K) over 100-cell sliding windows; Spearman correlation (ρ) is given in the legend. Boxplots show gene expression in uninfected cells (boxes: median and interquartile range; whiskers: 2.5 th and 97.5 th percentiles). See also G and S8H.

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Infection, Gene Expression, Virus, Ex Vivo, In Vivo

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet:

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Virus, Recombinant, Lysis, Electron Microscopy, Infection, Gene Expression, Sequencing, Software

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells). SMCs alone, with direct contact and without direct contact to isolated myenteric plexus cells.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Co-Culture Assay, Isolation

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Immunofluorescence staining of enteric nervous system (ENS) cells co-cultured with smooth muscle cells in a three-dimensional HyStem-C hydrogel, showing ( a ) glial fibrillary acidic protein (GFAP)–positive glial fibers (magenta), ( b ) smooth muscle cells stained for smooth muscle actin (SMA, cyan), ( c ) neuronal fibers labeled with βIII-tubulin (Tuj1, green), and ( d ) merged image. Cell nuclei are counterstained with DAPI (blue). Non-specific staining observed in the SMA channel indicates the presence of additional cell types, likely fibroblasts. Scale bar 50 μm. ( e ) higher-magnification image of a GFAP-positive glial cell (magenta). Scale bar 100 μm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Immunofluorescence, Staining, Cell Culture, Labeling

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Contracting muscle fibers in the live cultures of muscle cells together with ENS cells in HyStem-C Hydrogel (real time): ( a ) thin muscle fibers, ( b ) thick muscle fibers. Light microscopy.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Light Microscopy

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Smooth muscle and ENS cells in 3D scaffolds (14 days) co-cultured in 3D scaffold, showing neurons in green (ß-Tubulin III), muscle cells in red (smooth muscle actin (SMA)) and nuclei in blue (DRAQ5) ( a ) Confocal microscopy lower Magnification, ( b ) Confocal microscopy 3D structure in merged channels view from the side in higher magnification (Scale bars 50 μm) and ( c ) Confocal microscopy, separated channels view from above in higher magnification, ( d ) Electron microscopy of two muscle cells in close contact within the thickness of the three-dimensional matrix. The cells have large nuclei, an elongated shape, and actin microfilaments typical for muscle cells. Scale bar 2 μm, ( e ) Electron microscopy of plasma membrane of a muscle cell, with arrows indicating caveolae. Scale bar 200 nm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Cell Culture, Confocal Microscopy, Electron Microscopy, Clinical Proteomics, Membrane

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Images of 3 weeks old co-culture of ENS cells and muscle cells in different layouts: ( a ) SMCs alone, ( b ) paracrine interaction between SMCs and ENS cells, ( c ) direct contact between SMCs and ENS cells Scale bars 100 μm, ( d ) paracrine interaction between SMCs and ENS in higher magnification, ( e ) Direct contact between smooth muscle and ENS cells in higher magnification.– muscle cells Scale bars 20 μm, ( f ) 3D reconstruction of muscle fibers in confocal microscopy by the direct contact between SMCs and ENS, ( j ) neurons, which are intercommunicated in the neuronal net within the muscle layer by the direct contact between SMCs and ENS. Confocal microscopy, Green (ß-Tubulin III) – neurons, red (smooth muscle actin (SMA)) – muscle cells. Scale bars 50 μm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Co-Culture Assay, Confocal Microscopy

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Imaging of the whole thickness of the 3D scaffolds after 3 weeks of ENS cells and muscle cells co-culture in different layouts: ( a ) SMCs without ENS cells, ( b ) paracrine interaction between SMCs and ENS cells, ( c ) direct contact between SMCs and ENS cells. Confocal microscopy, Green (ß-Tubulin III) – neurons, red (smooth muscle actin (SMA)) – muscle cells.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Imaging, Co-Culture Assay, Confocal Microscopy

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Confocal microscopy images of smooth muscle and ENS cells co-cultured in 3D scaffolds (14 days of culture). Secretory vesicles and synapses are labelled in green (Synaptobrevin 2), while muscle cells in red (smooth muscle actin (SMA)). Scale bars 20 μm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Confocal Microscopy, Cell Culture

Journal: Advanced Science

Article Title: Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1

doi: 10.1002/advs.202307556

Figure Lengend Snippet: Celastrol improved neuronal mitochondrial dysfunction induced by ICH. A) The chemical structure of celastrol labeled with biotin is illustrated. B) Neurons were treated with 50 n m biotin or 50 n m Biotin‐Cel for 6 h, followed by immunofluorescence analysis using an anti‐biotin antibody (green) in neurons and mitotracker staining for mitochondria (red). Nuclei were stained with DAPI (blue). Representative images from triplicate experiments are shown. Arrows indicate the co‐localization of celastrol and mitochondria. Scale bar: 10 µm. C) Transmission electron microscopy of mitochondrial structures in neuronal cells of mice across different experimental groups. Scale bar: 5 µm. The neurons were stimulated with 10 µ m OxyHb for 12 h and subsequently exposed to varying doses of celastrol treatment for 24 h. Afterward, the cells were collected for assessment of mitochondrial function. D) Oxygen consumption rates (OCR) were measured by using the Seahorse XF‐24 Extracellular Flux Analyze, n = 3. E) Extracellular acidification rate (ECAR) was measured by using the Seahorse XF‐24 Extracellular Flux Analyze, n = 3. F) The ATP content was measured using a chemiluminescence assay, n = 6. G) Neurons from various experimental groups were subjected to JC‐1 staining and subsequently visualized under a Nikon fluorescence microscope. Scale bar: 100 µm. H) Neurons from different experimental groups were subjected to JC‐1 staining, and the fluorescence intensity was quantified using a fluorescent microplate reader with excitation/emission wavelengths of 514/529 nm for monomers and 585/590 nm for aggregates, n = 6. I) Cytoplasm and mitochondria were separated to detect the protein levels of Cyto C, which was normalized to 1.0 based on the mean value of each protein in the control group. β‐Tubulin and voltage‐dependent anion‐selective channel protein 1 (VDAC1) served as loading controls. J) Neurons from various experimental groups were lysed, and western blot analysis was performed to determine the protein levels of caspase‐9. The mean value of each protein in the control group was normalized to 1.0, with GAPDH serving as the loading control, n = 6. K) Neurons from various experimental groups were lysed, and western blot analysis was performed to determine the protein levels of caspase‐3. The mean value of each protein in the control group was normalized to 1.0, with β‐Tubulin serving as the loading control, n = 6. All data are presented as mean ± SD. Statistical significance was determined using one‐ or two‐way ANOVA with Tukey's multiple comparisons tests ( * p < 0.05, ** p < 0.001, *** p < 0.0001 vs control group; # p < 0.05, ## p < 0.001, ### p < 0.0001 vs vehicle group).

Article Snippet: Mitochondria were extracted using the Tissue and Cell Mitochondrial Extraction Kit (Beyotime, C3606, C3601) and separated from the cytoplasm through differential centrifugation of fresh brain tissue (processed within 1 h) and neuronal cells according to the manufacturer's instructions.

Techniques: Labeling, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy, Chemiluminescence Immunoassay, Fluorescence, Microscopy, Control, Western Blot

Journal: Advanced Science

Article Title: Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1

doi: 10.1002/advs.202307556

Figure Lengend Snippet: EPAC‐1 contributes to mitochondrial dysfunction induced by ICH through its interaction with VDAC1. A) Samples of cortex surrounding the hematoma in mice were collected at 3, 6, 12, 24, and 48 h post‐ICH induction. The protein levels of EPAC‐1 were assessed via western blot analysis, and the alterations in expression were quantified. β‐Tubulin served as loading controls, with a sham group being normalized to a value of 1.0 for accurate comparisons ( n = 6). B) EPAC‐1 activation was assessed by measuring the levels of activated Rap1‐GTP using a Rap1 Activation Assay Kit. β‐Tubulin served as loading controls, with each sham group being normalized to a value of 1 for accurate comparisons n = 6. C) Neurons underwent a 12 h stimulation with 10 µ m OxyHb. Subsequently, mitochondria and cytoplasm were isolated, and the protein levels of EPAC‐1 in both compartments were evaluated using western blot analysis. β‐Tubulin and Tom20 served as loading controls, with each control group being normalized to a value of 1 for accurate comparisons, n = 6. D) Protein‐protein interaction (PPI) network was queried from the STRING database ( https://string‐db.org ), followed by functional protein enrichment analysis conducted through Cytoscape and ClueGO. E) Rigid protein‐protein docking (ZDOCK) was conducted to investigate the relationship between EPAC‐1 and VDAC1. The PDB format of the protein structural domain was obtained from the Protein Data Bank (PDB) at http://www.rcsb.org/ . The ZDOCK module was used to identify docking sites and calculate ZDOCK scores. F) Cortex samples surrounding the hematoma in mice were collected from sham and ICH 24 h groups, followed by co‐immunoprecipitation to validate the interaction between EPAC‐1 and VDAC‐1. G,H) Following stimulation with 10 µ m OxyHb for 12 h, neurons were treated with the EPAC‐1 inhibitor ESI09 (10 µ m ) or activator 8CPT (10 µ m ) for 24 h. The levels of activated Rap1‐GTP were detected using a Rap1 Activation Assay Kit (G), while EPAC‐1 protein levels in both compartments were assessed via western blot analysis (H). Co‐immunoprecipitation was used to detect the interaction between EPAC‐1 and VDAC‐1 I), and mitochondrial structures were examined by means of transmission electron microscopy with a scale bar of 5 µm J). The ATP content was quantified L), and mitochondrial membrane potential (MMP) was measured by JC‐1 staining K). All data are presented as mean ± SD. Statistical significance was determined using one‐ or two‐way ANOVA with Tukey's multiple comparisons tests ( n = 6, *** p < 0.0001 vs control/ sham group; ### p < 0.0001 vs Vehicle group).

Article Snippet: Mitochondria were extracted using the Tissue and Cell Mitochondrial Extraction Kit (Beyotime, C3606, C3601) and separated from the cytoplasm through differential centrifugation of fresh brain tissue (processed within 1 h) and neuronal cells according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Activation Assay, Isolation, Control, Functional Assay, Protein Enrichment, Immunoprecipitation, Transmission Assay, Electron Microscopy, Membrane, Staining

Journal: Advanced Science

Article Title: Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1

doi: 10.1002/advs.202307556

Figure Lengend Snippet: Celastrol inhibits the elevation of EPAC‐1 activity and MPTP opening induced by ICH. A) Samples of cortex surrounding the hematoma in mice were collected 48 h after ICH induction and treatment with 1, 2, and 4 mg k −1 g celastrol. EPAC‐1 protein levels were detected by western blot. β‐Tubulin served as loading controls, with a sham group being normalized to a value of 1 for accurate comparisons, n = 6. B) After being stimulated with OxyHb, the neurons were treated with the EPAC‐1 inhibitor ESI09 (10 µ m ) or celastrol at concentrations of 25 and 50 n m . The activation levels of Rap1‐GTP were measured using a Rap1 Activation Assay Kit. β‐Tubulin served as loading controls, with each sham group being normalized to a value of 1 for accurate comparisons, n = 6. C) Following stimulation with OxyHb, the neurons were treated with 50 n m celastrol. Immunofluorescence analysis was performed using an anti‐EPAC‐1 antibody (green) to label neurons and mitotracker staining for mitochondria (red). Nuclei were counterstained with DAPI (blue). Representative images from triplicate experiments are presented, showing co‐localization of celastrol and mitochondria in the proximal neurite, as indicated by arrows. Scale bar: 10 µm. D) The protein levels of EPAC‐1 in the mitochondria and cytoplasm were evaluated by western blot analysis across different groups. The mean value of each protein in the control group was normalized to 1.0, with β‐tubulin and Tom 20 serving as the loading control, n = 6. E) Co‐immunoprecipitation was used to detect the interaction between EPAC‐1 and VDAC‐1. F) Mitochondria were pretreated with celastrol or ESI09 for 30 min, followed by exposure to Ca 2+ for another 10 min. Mitochondria swelling traces were recorded based on the absorbance at 540 nm. The mitochondrial swelling was quantified, n = 6. The neurons were stimulated with 10 µ m OxyHb for 12 h and subsequently exposed to varying doses of celastrol treatment for 24 h. Afterward, the cells were collected for assessment of mitochondrial function. G) Mitochondrial structures in neurons, with or without celastrol treatment following OxyHb stimulation, were observed using transmission electron microscopy. The scale bar was set at 5 µm. H) Neurons from various experimental groups were subjected to MitoSOX staining and subsequently visualized under a Nikon fluorescence microscope. I) Neurons from various experimental groups were subjected to MitoSOX staining, and the fluorescence intensity was quantified using a fluorescent microplate reader with excitation/emission wavelengths of 396/610 nm, n = 6. J) The Fluo3‐AM staining was used to detect calcium in the cytoplasm of neurons, which was subsequently visualized using a Nikon fluorescence microscope. K) The fluorescence intensity of Fluo3‐AM was measured using a fluorescent microplate reader with excitation and emission wavelengths set at 506 and 526 nm, respectively, n = 6. All data are presented as mean ± SD. Statistical significance was determined using one‐or two‐way ANOVA with Tukey's multiple comparisons tests ( * p < 0.05, ** p < 0.001, *** p < 0.0001 vs control group; # p < 0.05, ## p < 0.001, ### p < 0.0001 vs vehicle group).

Article Snippet: Mitochondria were extracted using the Tissue and Cell Mitochondrial Extraction Kit (Beyotime, C3606, C3601) and separated from the cytoplasm through differential centrifugation of fresh brain tissue (processed within 1 h) and neuronal cells according to the manufacturer's instructions.

Techniques: Activity Assay, Western Blot, Activation Assay, Immunofluorescence, Staining, Control, Immunoprecipitation, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy

Journal: Advanced Science

Article Title: Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1

doi: 10.1002/advs.202307556

Figure Lengend Snippet: Graphic illustration of neuroprotective effects and mechanisms of celastrol. Following ICH, EPAC‐1 is activated within neurons and translocated to the outer membrane of mitochondria. Here, it can form a complex with VDAC1, promoting MPTP opening and subsequent collapse of mitochondrial membrane potential. This results in Ca 2+ release, which induces neuronal apoptosis via cytochrome C (Cyto C). As a natural compound, celastrol can directly localize in mitochondria and interact with EPAC‐1 to modulate its activation, thereby impeding the binding of EPAC‐1 to VADC1. This ameliorates mitochondrial impairment in neuronal cells and exerts neuroprotective effects after ICH.

Article Snippet: Mitochondria were extracted using the Tissue and Cell Mitochondrial Extraction Kit (Beyotime, C3606, C3601) and separated from the cytoplasm through differential centrifugation of fresh brain tissue (processed within 1 h) and neuronal cells according to the manufacturer's instructions.

Techniques: Membrane, Activation Assay, Binding Assay