Journal: eLife

Article Title: A novel mouse model for LAMA2 -related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

doi: 10.7554/eLife.94288

Figure Lengend Snippet: ( A ) UMAP visualization of all the cells colored by sample. ( B ) UMAP visualization of all the cells from dy H /dy H and WT brains colored by cluster identity. ( C ) Cell composition proportion of every annotated cell cluster in dy H /dy H and WT separately. ( D ) Violin plots of the expression of Lama2 gene in WT and dy H /dy H in each annotated cell cluster. ( E ) Immunofluorescence of laminin α2, GFAP (astrocytes), and PLP1 on P14 mouse brain slices. DAPI was for nuclei staining. White arrows indicated the cortical surface. ( F ) Gene Ontology (GO) enrichment of differentially expressed genes in vascular and leptomeningeal fibroblasts, vascular smooth muscle cells, and astrocytes. ( G ) Cell-cell communications among cell clusters. Total number of inferred cell-cell interaction counts (left) and strength (right) in dy H /dy H and WT, respectively. Heatmap of ligand-receptor interaction counts in all pairwise cell clusters in WT and dy H /dy H brains.

Article Snippet: Immunofluorescence was performed according to standard procedures with antibodies against the N-terminus of the laminin α2 chain (rat monoclonal, 4H8-2, Sigma, Saint Louis, USA), the laminin α1 chain (rat monoclonal, MAB4656, R&D Systems, Minneapolis, USA), myogenic differentiation antigen 1 (MYOD1) (mouse monoclonal, ab16148, Abcam, Cambridge, UK), myogenin (MYOG) (mouse monoclonal, MAB66861, R&D Systems, Minneapolis, USA), myogenic factor 5 (MYF5) (mouse monoclonal, MAB4027, R&D Systems, Minneapolis, USA), myosin heavy chain (MYHC) (mouse monoclonal, MAB4470, R&D Systems, Minneapolis, USA), desmin (mouse monoclonal, MA5-15306, Invitrogen, CA), CD68 (rabbit IgG, BA3638, Boster, CA), and clone IIH6C4 for α-DG (mouse monoclonal, 05–593, Merck Millipore, Darmstadt, Germany).

Techniques: Expressing, Immunofluorescence, Staining

Journal: eLife

Article Title: A novel mouse model for LAMA2 -related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

doi: 10.7554/eLife.94288

Figure Lengend Snippet: ( A ) The ligand-receptor pairs in laminin pathway. ( B ) Violin plots showed the expression of Mbp , Mobp , Plp1 , Slc1a2 , Mt3 , and Slc6a13 in every annotated cell cluster in dy H /dy H and wild-type (WT) separately. *** for p-value <0.001, ** for p-value <0.01, * for p-value <0.05. ( C ) Hypothesis that cortical dysplasia (cobblestone malformation) and neuronal over-migration in the cortex due to a defective gliovascular basal lamina of the blood-brain barrier caused by the lack of laminin α2 in LAMA2 -MD.

Article Snippet: Immunofluorescence was performed according to standard procedures with antibodies against the N-terminus of the laminin α2 chain (rat monoclonal, 4H8-2, Sigma, Saint Louis, USA), the laminin α1 chain (rat monoclonal, MAB4656, R&D Systems, Minneapolis, USA), myogenic differentiation antigen 1 (MYOD1) (mouse monoclonal, ab16148, Abcam, Cambridge, UK), myogenin (MYOG) (mouse monoclonal, MAB66861, R&D Systems, Minneapolis, USA), myogenic factor 5 (MYF5) (mouse monoclonal, MAB4027, R&D Systems, Minneapolis, USA), myosin heavy chain (MYHC) (mouse monoclonal, MAB4470, R&D Systems, Minneapolis, USA), desmin (mouse monoclonal, MA5-15306, Invitrogen, CA), CD68 (rabbit IgG, BA3638, Boster, CA), and clone IIH6C4 for α-DG (mouse monoclonal, 05–593, Merck Millipore, Darmstadt, Germany).

Techniques: Expressing, Migration

Journal: eLife

Article Title: A novel mouse model for LAMA2 -related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

doi: 10.7554/eLife.94288

Figure Lengend Snippet: ( A ) Quantitative analysis of immunofluorescence staining for myosin heavy chain (MYHC), MYOG, desmin, MYOD1, β-tubulin, and MYF-5 in wild-type (WT) and dy H /dy H muscles at P14. ( B ) Quantitative analysis of Western blot for laminin α2, F-actin, α-actin, MYH2, MYHC, desmin, β-tubulin, MYOG, and MYOD1 in P14 WT and dy H /dy H muscles. *** for p-value <0.001, ** for p-value <0.01, * for p-value <0.05.

Article Snippet: Immunofluorescence was performed according to standard procedures with antibodies against the N-terminus of the laminin α2 chain (rat monoclonal, 4H8-2, Sigma, Saint Louis, USA), the laminin α1 chain (rat monoclonal, MAB4656, R&D Systems, Minneapolis, USA), myogenic differentiation antigen 1 (MYOD1) (mouse monoclonal, ab16148, Abcam, Cambridge, UK), myogenin (MYOG) (mouse monoclonal, MAB66861, R&D Systems, Minneapolis, USA), myogenic factor 5 (MYF5) (mouse monoclonal, MAB4027, R&D Systems, Minneapolis, USA), myosin heavy chain (MYHC) (mouse monoclonal, MAB4470, R&D Systems, Minneapolis, USA), desmin (mouse monoclonal, MA5-15306, Invitrogen, CA), CD68 (rabbit IgG, BA3638, Boster, CA), and clone IIH6C4 for α-DG (mouse monoclonal, 05–593, Merck Millipore, Darmstadt, Germany).

Techniques: Immunofluorescence, Staining, Muscles, Western Blot

Journal: eLife

Article Title: A novel mouse model for LAMA2 -related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

doi: 10.7554/eLife.94288

Figure Lengend Snippet: Colocalization of α-dystroglycan (red fluorescence, blue arrow) and laminin α2 chain (green fluorescence, yellow arrow) showed that α-dystroglycan protein was normally located and expressed in the cell membrane both in the wild-type (WT) and dy H /dy H muscles. Scale bars: 40 µm.

Article Snippet: Immunofluorescence was performed according to standard procedures with antibodies against the N-terminus of the laminin α2 chain (rat monoclonal, 4H8-2, Sigma, Saint Louis, USA), the laminin α1 chain (rat monoclonal, MAB4656, R&D Systems, Minneapolis, USA), myogenic differentiation antigen 1 (MYOD1) (mouse monoclonal, ab16148, Abcam, Cambridge, UK), myogenin (MYOG) (mouse monoclonal, MAB66861, R&D Systems, Minneapolis, USA), myogenic factor 5 (MYF5) (mouse monoclonal, MAB4027, R&D Systems, Minneapolis, USA), myosin heavy chain (MYHC) (mouse monoclonal, MAB4470, R&D Systems, Minneapolis, USA), desmin (mouse monoclonal, MA5-15306, Invitrogen, CA), CD68 (rabbit IgG, BA3638, Boster, CA), and clone IIH6C4 for α-DG (mouse monoclonal, 05–593, Merck Millipore, Darmstadt, Germany).

Techniques: Fluorescence, Membrane, Muscles

Journal: eLife

Article Title: A novel mouse model for LAMA2 -related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

doi: 10.7554/eLife.94288

Figure Lengend Snippet: Laminin α2 deficiency in the basement membrane due to pathogenic variants in the LAMA2 gene leads to loss of dystroglycan/integrin-matrix scaffolds and extracellular matrix networks. Then, a series of molecular changes associated with the cytoskeleton, extracellular matrix, fibrosis, inflammation, apoptosis, pyroptosis, and mitochondrial energy metabolism occur.

Article Snippet: Immunofluorescence was performed according to standard procedures with antibodies against the N-terminus of the laminin α2 chain (rat monoclonal, 4H8-2, Sigma, Saint Louis, USA), the laminin α1 chain (rat monoclonal, MAB4656, R&D Systems, Minneapolis, USA), myogenic differentiation antigen 1 (MYOD1) (mouse monoclonal, ab16148, Abcam, Cambridge, UK), myogenin (MYOG) (mouse monoclonal, MAB66861, R&D Systems, Minneapolis, USA), myogenic factor 5 (MYF5) (mouse monoclonal, MAB4027, R&D Systems, Minneapolis, USA), myosin heavy chain (MYHC) (mouse monoclonal, MAB4470, R&D Systems, Minneapolis, USA), desmin (mouse monoclonal, MA5-15306, Invitrogen, CA), CD68 (rabbit IgG, BA3638, Boster, CA), and clone IIH6C4 for α-DG (mouse monoclonal, 05–593, Merck Millipore, Darmstadt, Germany).

Techniques: Membrane