gene cell matrices counts  (10X Genomics)

Journal: bioRxiv

Article Title: Mgat4b mediated selective N -glycosylation regulates melanocyte development and melanoma progression

doi: 10.1101/2024.10.10.617552

Figure Lengend Snippet: A. UMAP visualization of the Zebrafish Sox10+ve cells colored by the identified states B. Dot plot showing the top marker genes enriched in each cluster with the size showing the percent of cell expressing the gene and color showing the scaled mean expression value in each cluster (Wilcoxon-Mann-Whitney test with average log fold change > 0.25 and adjusted p value ≤ 0.05). List includes known cell-type marker genes (marked in red box) and new candidate markers C. UMAP plot shows enrichment of mgat4b in pigment progenitor and melanophore clusters D. Mammalian protein N -glycosylation starts in the endoplasmic reticulum and continues in the Golgi apparatus, where three mature types of N -glycan structures—high-mannose, hybrid, and complex—are formed. The enzyme GT-4b transfers N -acetylglucosamine in a β1→4 linkage to α1→3-linked mannose, which kickstarts the creation of a specific N-glycan branch. This branch often includes a sialylated N -acetyllactosamine sequence, highlighting its distinctive features in glycan structure E. Heatmap of NicheNetR-identified ligand/receptor pairs indicating interaction potential between mgat4b +ve receiver cells and other sender cells sampled by scRNAseq of zebrafish Sox10+ve cells.

Article Snippet: The Gene-cell matrices (Control: 1923, mgat4b knockout: 4120) were uploaded on RStudio (R version 4.3)( ) and standard quality control metrics with the Seurat package (v.5.1) ( ).

Techniques: Marker, Expressing, MANN-WHITNEY, Sequencing

Journal: bioRxiv

Article Title: Mgat4b mediated selective N -glycosylation regulates melanocyte development and melanoma progression

doi: 10.1101/2024.10.10.617552

Figure Lengend Snippet: A. UMAP visualization of the zebrafish Mitfa+ve cells integrated from Non-targeted control (NTC) and melanocyte specific knock out of mgat4b (m4b mut)cells colored by the identified states B . Dot plot showing the top marker genes enriched in each cluster with the size showing the percent of cell expressing the gene and color showing the scaled mean expression value in each cluster C . UMAPs of Mitfa+ cells with color change from light pink (negative) to red based on log normalized scaled expression of mitfa, twist1a, elavl3, crestin, aox5, anxa1a and tyrp1b and foxd3 D . UMAP visualization of the Zebrafish Mitfa+ve cells from NTC and m4b mut cells colored by the identified states E . Heat map showing the proportion of cells belonging to certain cell-type distributed between NTC and m4b mut samples F . Waterfall plot depicting top upregulated GO-terms in MIX+ cluster plotted against their normalized enrichment score G . Pseudotime ordering of the cells, coloring based on pseudotime scores (Wilcoxon-Mann-Whitney test with average log fold change > 1 and adjusted p value ≤ 0.05)

Article Snippet: The Gene-cell matrices (Control: 1923, mgat4b knockout: 4120) were uploaded on RStudio (R version 4.3)( ) and standard quality control metrics with the Seurat package (v.5.1) ( ).

Techniques: Control, Knock-Out, Marker, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Mgat4b mediated selective N -glycosylation regulates melanocyte development and melanoma progression

doi: 10.1101/2024.10.10.617552

Figure Lengend Snippet: A. Brightfield images shows wildtype control and Mgat4b knockout cells morphology, Scale bar-100 µm B . Western blot shows protein levels of Mgat4b in wildtype control and two clones of Mgat4b KO (Mut1 and Mut2) along with Cas9 control in B16-mouse melanoma cells C. Phalloidin staining shows actin filament distribution in wildtype control and Mgat4 b knockout (Mut1) cells, Scale bar-75 µm D. Toluidine blue labels cells on Matrigel depicting the invasion capability of wildtype control and Mgat4b knockout(Mut1) cells, Scale bar-50 µm E. Bar graph depicting percentage of invasion of wildtype control and Mgat4b knockout cells (Mut1) F, G. Rose plot depicting spatial distribution of wildtype control and Mgat4b knockout cells(Mut1) in chemotaxis chamber assay H. Xenograft of mouse melanoma cells (wildtype control and Mgat4b knockout(Mut1) cells) into the 2dpf zebrafish yolk I. Bar plot shows relative change in tumor area of wildtype control and Mgat4b knockout(Mut1) cells, Scale bar-50 µm J. Bar plot shows number of tumor cells migrating out of primary cluster to invade neighboring tissues per frame K. Phalloidin staining shows actin filament distribution in Mgat4b KO(Mut1) cells complemented with Empty vector (EV), MGAT4B and MGAT4A protein, Scale bar-50µm L. Brightfield images showing cells morphology and colony formation in Mgat4b KO(Mut1) cells complemented with EV, MGAT4B and MGAT4A protein along with wildtype and Mgat4b KO cells (No transfection control), the numbers listed under each image indicate how many out of 35 cells exhibit the shown phenotype, Scale bar-175 µm *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 and ns P > 0.05

Article Snippet: The Gene-cell matrices (Control: 1923, mgat4b knockout: 4120) were uploaded on RStudio (R version 4.3)( ) and standard quality control metrics with the Seurat package (v.5.1) ( ).

Techniques: Control, Knock-Out, Western Blot, Clone Assay, Staining, Chemotaxis Assay, Boyden Chamber Assay, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: Mgat4b mediated selective N -glycosylation regulates melanocyte development and melanoma progression

doi: 10.1101/2024.10.10.617552

Figure Lengend Snippet: A. Venn diagram depicting number of proteins identified by mass spectrometry analysis of the DSL-enriched fractions of Wildtype and Mgat4b KO proteins C. List of differentially enriched proteins in wildtype lysates with respect to Mgat4b KO along with their Uniprot ID, Sum PEP Score, PSMs, calc. PI and their functional profiling and predicted N -glycosylation sites by NetNGlyc -1.0 database D. Western blot analysis of Tyrp1, Kit, Gpnmb and Jup in wildtype and Mgat4b KO lysate along with PNGase treated wildtype lysate E. Western blot analysis of Tyrp1, c-Kit, Gpnmb , and Jup following biotinylated DSL pull-down in wildtype and Mgat4b knockout lysates F. Confocal image of immunocytochemistry using Tyrp1, Kit, Gpnmb and Jup (red) on permeabilized wildtype and Mgat4b KO cells, counterstained by DAPI(blue). The overlay of both channels is depicted. Scale bars: 50 µm

Article Snippet: The Gene-cell matrices (Control: 1923, mgat4b knockout: 4120) were uploaded on RStudio (R version 4.3)( ) and standard quality control metrics with the Seurat package (v.5.1) ( ).

Techniques: Mass Spectrometry, Functional Assay, Western Blot, Knock-Out, Immunocytochemistry

Journal: bioRxiv

Article Title: Mgat4b mediated selective N -glycosylation regulates melanocyte development and melanoma progression

doi: 10.1101/2024.10.10.617552

Figure Lengend Snippet: A. Scatter plot depicting expression of MGAT4B in Skin cutaneous melanoma (SKCM) patients with respect to normal individuals, data procured from TCGA B. 60 days old Mazerati zebrafish displaying melanoblast-derived tumor developed by combinations of vectors expressing the oncogene BRAF V600E , targeting tp53 and ptena/b initiate melanoma left-Mitfa-BRAF V600E , Mitfa- p53 -/- , Mitfa- ptena/b -/- and Mitfa:EV, right-Mitfa: mgat4b -/- C. Melanoma-free survival curves of ASWT zebrafish injected with the indicated combinations of vectors expressing Mitfa-BRAF V600E , Mitfa- p53 -/- , Mitfa- ptena/b -/- and Mitfa:EV or Mitfa: mgat4b -/- D. Schematic illustration of the experimental approach adapted to isolate and enrich(percol based gradient) melanophores from one month old wildtype and Mazerati fishes E. PCA analysis plot depicting relationship between the three samples sequenced F. Euler plot depicting the number of differentially expressed genes in melanocytes/melanoma cells from Mazerati fishes (control and M4b mut melanoma) with respect to wildtype melanophores G. Dot plot depicting functional enrichment from the upregulated genes in control and M4b mut melanophores/melanoma cells with respect to wildtype melanophores H. Dot plot depicting upregulated and downregulated processes in M4b mut melanoma with respect to control melanophores/melanoma *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 and ns P > 0.05

Article Snippet: The Gene-cell matrices (Control: 1923, mgat4b knockout: 4120) were uploaded on RStudio (R version 4.3)( ) and standard quality control metrics with the Seurat package (v.5.1) ( ).

Techniques: Expressing, Derivative Assay, Injection, Control, Functional Assay