Journal: The Journal of Clinical Investigation

Article Title: Net39 protects muscle nuclei from mechanical stress during the pathogenesis of Emery-Dreifuss muscular dystrophy

doi: 10.1172/JCI163333

Figure Lengend Snippet: ( A ) H&E (top) and WGA staining (bottom) of Ctrl and cKO GP muscles at 5 months of age. Scale bar: 50 μm. ( B ) Myofiber area distribution from WGA-stained GP sections (left) and average myofiber area (right) from Ctrl (black) and cKO (red) mice at 5 months of age. Unpaired, 2-tailed Student’s t test. n = 3 mice. ( C ) Immunohistochemistry of Ctrl and cKO GP muscles at 5 months of age for NADH, COX, SDH, and metachromatic ATPase staining. Type I fibers stain darkly, and type II fibers stain lightly. Red arrowheads indicate small angular myofibers in cKO muscle. Scale bar: 50 μm. ( D ) Immunostaining for MYH7 and WGA (bottom panels) of Ctrl and cKO GP muscles at 5 months of age and cKO GP muscle at 9 months of age. The magnified area on the bottom panels shows the presence of small angular fibers positive for MYH7 in cKO GP. The same area in Ctrl GP is usually devoid of MYH7-positive fibers. Scale bars: 500 μm (top); 50 μm (bottom). ( E ) Immunostaining for type I (MYH7), type IIa (MYH2), and type IIb (MYH4) myofibers of Ctrl and cKO GP muscles at 5 months of age. The magnified area on the right shows the presence of small angular fibers positive for MYH7 or MYH2 in cKO GP. Scale bars: 500 μm (left); 50 μm (right). ( F ) Quantification of small angular fibers over the total number of fibers from WGA staining in D . One-way ANOVA followed by Tukey’s multiple-comparisons test. n = 4 mice. ( G ) Quantification of myofiber areas from WGA staining in D . Two-way ANOVA followed by Tukey’s multiple-comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. n = 4 mice. Approximately 60-90 fibers per mouse were quantified for B , F , and G . Data are represented as mean ± SEM.

Article Snippet: The following antibodies were used: SUN2 (MilliporeSigma, MABT880), MYH7 (Developmental Studies Hybridoma Bank [DSHB], BA-D5), MYH2 (DSHB, SC-71), MYH4 (DSHB, BF-F3), MYH3 (DSHB, F1.652-s), γH2A.X (Cell Signaling Technology, 9718), MEF2C (Cell Signaling Technology, 5030), CD11B (Abcam, ab133357), DESMIN (Agilent, M076029-2), cleaved caspase-3 (Cell Signaling Technology, 9661) and WGA (Thermo Fisher, W11261), laminin (MilliporeSigma, L9393), and DAPI (MilliporeSigma, D9542).

Techniques: Staining, Immunohistochemistry, Immunostaining

Journal: The Journal of Clinical Investigation

Article Title: Net39 protects muscle nuclei from mechanical stress during the pathogenesis of Emery-Dreifuss muscular dystrophy

doi: 10.1172/JCI163333

Figure Lengend Snippet: ( A ) Immunostaining for MYH7 (red) and WGA (green) in healthy Ctrl and EDMD patient muscle biopsies (left). White arrowheads indicate atrophic myofibers. Scale bar: 50 μm. Quantification of percentage of atrophic myofibers out of the total number of myofibers (right). ** P < 0.01. Approximately 60–100 myofibers per sample. ( B ) Immunostaining for γH2A.X (red), WGA (white), and DAPI (blue) in Ctrl and EDMD muscle biopsies. Scale bar: 50 μm (left). Quantification of γH2A.X-positive nuclei (right). **** P < 0.0001. Approximately 50–100 nuclei analyzed per sample. ( C ) Immunostaining for γH2A.X (red), MYH7 (green), WGA (white), and DAPI (blue) in EDMD muscle biopsies (left). γH2A.X is mostly detected in small angular myofibers that are also MYH7 positive. White arrowheads indicate γH2A.X-positive nuclei. An atrophic myofiber with γH2A.X-positive nuclei (red box) is enlarged (middle). Quantification of γH2A.X-positive fibers that are also atrophic among all atrophic myofibers in Ctrl and EDMD muscle (right). *** P < 0.001. Scale bars: 50 μm. Approximately 60–100 myofibers analyzed per sample. ( D ) Upstream regulator analysis of TFs for the most upregulated genes from a published human EDMD patient microarray ( , ). ( E ) Immunostaining of MEF2C (green), WGA (white), and DAPI (red) in Ctrl and EDMD muscle biopsies. White arrowheads indicate MEF2C-positive nuclei in EDMD. Scale bar: 10 μm. Quantification of MEF2C-positive nuclei (right). * P < 0.05. Approximately 50–100 nuclei analyzed per sample. Unpaired, 2-tailed Student’s t test was performed for A , B , C , and E . n = 3 human samples were analyzed for A , B , C , and E . Data are represented as mean ± SEM.

Article Snippet: The following antibodies were used: SUN2 (MilliporeSigma, MABT880), MYH7 (Developmental Studies Hybridoma Bank [DSHB], BA-D5), MYH2 (DSHB, SC-71), MYH4 (DSHB, BF-F3), MYH3 (DSHB, F1.652-s), γH2A.X (Cell Signaling Technology, 9718), MEF2C (Cell Signaling Technology, 5030), CD11B (Abcam, ab133357), DESMIN (Agilent, M076029-2), cleaved caspase-3 (Cell Signaling Technology, 9661) and WGA (Thermo Fisher, W11261), laminin (MilliporeSigma, L9393), and DAPI (MilliporeSigma, D9542).

Techniques: Immunostaining, Microarray

Journal: The Journal of Clinical Investigation

Article Title: Net39 protects muscle nuclei from mechanical stress during the pathogenesis of Emery-Dreifuss muscular dystrophy

doi: 10.1172/JCI163333

Figure Lengend Snippet: ( A ) H&E (top) and WGA staining (bottom) of Ctrl and cKO GP muscles at 5 months of age. Scale bar: 50 μm. ( B ) Myofiber area distribution from WGA-stained GP sections (left) and average myofiber area (right) from Ctrl (black) and cKO (red) mice at 5 months of age. Unpaired, 2-tailed Student’s t test. n = 3 mice. ( C ) Immunohistochemistry of Ctrl and cKO GP muscles at 5 months of age for NADH, COX, SDH, and metachromatic ATPase staining. Type I fibers stain darkly, and type II fibers stain lightly. Red arrowheads indicate small angular myofibers in cKO muscle. Scale bar: 50 μm. ( D ) Immunostaining for MYH7 and WGA (bottom panels) of Ctrl and cKO GP muscles at 5 months of age and cKO GP muscle at 9 months of age. The magnified area on the bottom panels shows the presence of small angular fibers positive for MYH7 in cKO GP. The same area in Ctrl GP is usually devoid of MYH7-positive fibers. Scale bars: 500 μm (top); 50 μm (bottom). ( E ) Immunostaining for type I (MYH7), type IIa (MYH2), and type IIb (MYH4) myofibers of Ctrl and cKO GP muscles at 5 months of age. The magnified area on the right shows the presence of small angular fibers positive for MYH7 or MYH2 in cKO GP. Scale bars: 500 μm (left); 50 μm (right). ( F ) Quantification of small angular fibers over the total number of fibers from WGA staining in D . One-way ANOVA followed by Tukey’s multiple-comparisons test. n = 4 mice. ( G ) Quantification of myofiber areas from WGA staining in D . Two-way ANOVA followed by Tukey’s multiple-comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. n = 4 mice. Approximately 60-90 fibers per mouse were quantified for B , F , and G . Data are represented as mean ± SEM.

Article Snippet: The following antibodies were used: SUN2 (MilliporeSigma, MABT880), MYH7 (Developmental Studies Hybridoma Bank [DSHB], BA-D5), MYH2 (DSHB, SC-71), MYH4 (DSHB, BF-F3), MYH3 (DSHB, F1.652-s), γH2A.X (Cell Signaling Technology, 9718), MEF2C (Cell Signaling Technology, 5030), CD11B (Abcam, ab133357), DESMIN (Agilent, M076029-2), cleaved caspase-3 (Cell Signaling Technology, 9661) and WGA (Thermo Fisher, W11261), laminin (MilliporeSigma, L9393), and DAPI (MilliporeSigma, D9542).

Techniques: Staining, Muscles, Immunohistochemistry, Immunostaining

Journal: The Journal of Clinical Investigation

Article Title: Net39 protects muscle nuclei from mechanical stress during the pathogenesis of Emery-Dreifuss muscular dystrophy

doi: 10.1172/JCI163333

Figure Lengend Snippet: ( A ) H&E (top) and WGA staining (bottom) of Ctrl and cKO GP muscles at 5 months of age. Scale bar: 50 μm. ( B ) Myofiber area distribution from WGA-stained GP sections (left) and average myofiber area (right) from Ctrl (black) and cKO (red) mice at 5 months of age. Unpaired, 2-tailed Student’s t test. n = 3 mice. ( C ) Immunohistochemistry of Ctrl and cKO GP muscles at 5 months of age for NADH, COX, SDH, and metachromatic ATPase staining. Type I fibers stain darkly, and type II fibers stain lightly. Red arrowheads indicate small angular myofibers in cKO muscle. Scale bar: 50 μm. ( D ) Immunostaining for MYH7 and WGA (bottom panels) of Ctrl and cKO GP muscles at 5 months of age and cKO GP muscle at 9 months of age. The magnified area on the bottom panels shows the presence of small angular fibers positive for MYH7 in cKO GP. The same area in Ctrl GP is usually devoid of MYH7-positive fibers. Scale bars: 500 μm (top); 50 μm (bottom). ( E ) Immunostaining for type I (MYH7), type IIa (MYH2), and type IIb (MYH4) myofibers of Ctrl and cKO GP muscles at 5 months of age. The magnified area on the right shows the presence of small angular fibers positive for MYH7 or MYH2 in cKO GP. Scale bars: 500 μm (left); 50 μm (right). ( F ) Quantification of small angular fibers over the total number of fibers from WGA staining in D . One-way ANOVA followed by Tukey’s multiple-comparisons test. n = 4 mice. ( G ) Quantification of myofiber areas from WGA staining in D . Two-way ANOVA followed by Tukey’s multiple-comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. n = 4 mice. Approximately 60-90 fibers per mouse were quantified for B , F , and G . Data are represented as mean ± SEM.

Article Snippet: The following antibodies were used: SUN2 (MilliporeSigma, MABT880), MYH7 (Developmental Studies Hybridoma Bank [DSHB], BA-D5), MYH2 (DSHB, SC-71), MYH4 (DSHB, BF-F3), MYH3 (DSHB, F1.652-s), γH2A.X (Cell Signaling Technology, 9718), MEF2C (Cell Signaling Technology, 5030), CD11B (Abcam, ab133357), DESMIN (Agilent, M076029-2), cleaved caspase-3 (Cell Signaling Technology, 9661) and WGA (Thermo Fisher, W11261), laminin (MilliporeSigma, L9393), and DAPI (MilliporeSigma, D9542).

Techniques: Staining, Muscles, Immunohistochemistry, Immunostaining