Journal: Life Science Alliance

Article Title: The LINC complex ensures accurate centrosome positioning during prophase

doi: 10.26508/lsa.202302404

Figure Lengend Snippet: (A) Representative immunofluorescence images of control and shSUN1-treated cells, immunostained for SUN1. (B) Representative immunofluorescence images of control and shSUN2-treated cells, immunostained for SUN2. Note the co-localization between tubulin and the SUN proteins in the merged image inset. (C, D) Representative immunofluorescence images of control and shSUN1-treated RPE-1 cells (C), or control and shSUN2-treated cells (D). Nocodazole (Noc) was added to the cells for 10 min, to induce microtubule depolymerization. Note the decrease in SUN1 and SUN2 staining between the centrosomes. (E, F, G) Representative frames from movies of control (E), shSUN1-treated (F), or shSUN2-treated (G) cells, stably expressing H2B-GFP and RFP-tubulin, seeded on fibronectin, during mitotic entry. Time is in sec. Time zero corresponds to nuclear envelope permeabilization (NEP). Yellow arrows indicate the centrosome position. (H, I, J) Correlation between the angle centrosome–centrosome (x-axis) and the angle centrosome–nucleus (y-axis) at the moment of NEP, for control ((H); n = 33), shSUN1-treated ((I); n = 33), and shSUN2-treated ((J); n = 31) cells. (K, L, M) Polar plots quantifying centrosome positioning relative to the longest nuclear axis at NEP for control (K), shSUN1-treated ((L); * P = 0.0165), and shSUN2-treated ((M); * P = 0.0175) cells. Yellow arrowheads indicate centrosome positions. All scale bars, 10 μm.

Article Snippet: The primary antibodies used were anti-lamin A (C-terminal; 1:1,000; L1293; Sigma-Aldrich), anti-SUN1 (1:500; MABT892; Merck Millipore), anti-SUN2 (1:500; MABT880; Merck Millipore), anti-GAPDH (1:20,000; 60004-1-Ig; Proteintech), and anti-β-tubulin (1:5,000; Ab6046; Abcam).

Techniques: Immunofluorescence, Staining, Stable Transfection, Expressing

Journal: Life Science Alliance

Article Title: The LINC complex ensures accurate centrosome positioning during prophase

doi: 10.26508/lsa.202302404

Figure Lengend Snippet: (A) Representative images of control and shSUN1-treated cells, stably expressing H2B-GFP and RFP-tubulin seeded on fibronectin and immunostained for SUN1. (B) Western blot to confirm the efficiency of SUN1 depletion in the overall population. (C) Representative images of control and shSUN2-treated cells, stably expressing H2B-GFP and RFP-tubulin seeded on fibronectin, immunostained for SUN2. (D) Western blot to confirm the efficiency of SUN2 depletion in the overall population. Yellow arrows indicate cells with higher depletion levels. (E) Expression of the ∆PPPL-KASH (top panel) and DN-KASH (bottom panel) constructs in RPE-1 prophase cells. Yellow arrows indicate the NE. (F) Representative image of a control RPE-1 cell in prophase, immunostained for nesprin-1 and nesprin-2, highlighting their NE localization. For all images, scale bars represent 10 mm. (G) Representative immunofluorescence images from ∆PPPL-KASH (top panel) and DN-KASH (bottom panel), to highlight LMNB1 localization. (H) Quantification of LMNB1 levels between ∆PPPL-KASH and DN-KASH (n.s., not significant). Source data are available for this figure.

Article Snippet: The primary antibodies used were anti-lamin A (C-terminal; 1:1,000; L1293; Sigma-Aldrich), anti-SUN1 (1:500; MABT892; Merck Millipore), anti-SUN2 (1:500; MABT880; Merck Millipore), anti-GAPDH (1:20,000; 60004-1-Ig; Proteintech), and anti-β-tubulin (1:5,000; Ab6046; Abcam).

Techniques: Stable Transfection, Expressing, Western Blot, Construct, Immunofluorescence

Journal: Life Science Alliance

Article Title: The LINC complex ensures accurate centrosome positioning during prophase

doi: 10.26508/lsa.202302404

Figure Lengend Snippet: (A) Representative immunofluorescence images of a ∆PPPL-KASH (top panel) and a DN-KASH (bottom panel) cell, immunostained for nesprin-2 (SYNE2). Note how the expression of DN-KASH displaces nesprin-2 from the NE (yellow arrows), as opposed to the expression of ∆PPPL-KASH. (B, E) Representative frames from control ((B); ∆PPPL-KASH) and DN-KASH (E)–treated cells, stably expressing tubulin-GFP and treated with SiR-DNA, seeded on fibronectin, during mitotic entry. Yellow arrowheads indicate centrosome position. (C, F) Polar plots quantifying centrosome positioning relative to the longest nuclear axis at nuclear envelope permeabilization (NEP) for RPE-1 expressing ∆PPPL-KASH ((C); n = 30) or DN-KASH ((F); n = 29; * P = 0.0237) cells. (D, G) Correlation between the angle centrosome–centrosome (x-axis) and the angle centrosome–nucleus (y-axis) at the moment of NEP for control ((D); ∆PPPL-KASH) or DN-KASH–expressing (G) cells. (H) Representative frame from a movie of a DN-KASH–expressing cell, stably expressing tubulin-GFP and treated with SiR-DNA, highlighting centrosome detachment from the NE (white arrowheads). (I) Quantification of centrosome displacement from the NE during mitotic entry for ∆PPPL-KASH– or DN-KASH–expressing cells before (−300 s) and at NEP. (J) Representative immunofluorescence images of control (top panel), ∆PPPL-KASH (middle panel), or DN-KASH (bottom panel) cells, labelled for the dynein adaptor, dynactin-1. Please note the decreased dynactin-1 signal at the NE of DN-KASH–expressing cells, when compared to control or ∆PPPL-KASH cells (yellow arrowheads). Red arrowheads indicate centrosomes positioned above and beneath the nucleus (lateral projections). (K, L) Quantification of the normalized fluorescence intensity signal of dynactin-1 signal on the NE ((K); *** P < 0.001) and cytoplasm ((L); P = 0.079) of ∆PPPL-KASH (n = 36) or DN-KASH (n = 35) cells. (M) Representative immunofluorescence images of control (top panel), SUN1 depleted (middle panel) and SUN2 depleted (bottom panel) RPE-1 cells, showing dynactin-1 accumulation on the NE. Red arrowheads indicate centrosomes. (N) Quantification of dynactin-1 levels on the NE following CDK1i washout for the different experimental groups. ** P = 0.086, **** P < 0.0001. Time is in sec, and time zero corresponds to NEP. Scale bars, 10 μm.

Article Snippet: The primary antibodies used were anti-lamin A (C-terminal; 1:1,000; L1293; Sigma-Aldrich), anti-SUN1 (1:500; MABT892; Merck Millipore), anti-SUN2 (1:500; MABT880; Merck Millipore), anti-GAPDH (1:20,000; 60004-1-Ig; Proteintech), and anti-β-tubulin (1:5,000; Ab6046; Abcam).

Techniques: Immunofluorescence, Expressing, Stable Transfection, Fluorescence

Journal: Life Science Alliance

Article Title: The LINC complex ensures accurate centrosome positioning during prophase

doi: 10.26508/lsa.202302404

Figure Lengend Snippet: (A) Representative immunofluorescence images showing the NE localization of nesprin-1 (SYNE1) and nesprin-2 (SYNE2) for RPE-1 (top panel), U2-OS (middle panel), and MDA-MB-468 (bottom panel) cell lines. (B, C, D, E) Quantification of the levels of SYNE1 (B), SYNE2 (C), SUN1 (D), and SUN2 (E) obtained from immunofluorescence images for all cell lines (**** P < 0.001; ** P < 0.01; and n.s., not significant). (F) Representative immunofluorescence images showing the NE localization of dynactin-1 in RPE-1 (top panel), U2-OS (middle panel), and MDA-MB-468 (bottom panel). (G) Quantification of the levels of dynactin-1 on the NE obtained from immunofluorescence images for all cell lines (**** P < 0.0001). Scale bars for all images, 10 mm.

Article Snippet: The primary antibodies used were anti-lamin A (C-terminal; 1:1,000; L1293; Sigma-Aldrich), anti-SUN1 (1:500; MABT892; Merck Millipore), anti-SUN2 (1:500; MABT880; Merck Millipore), anti-GAPDH (1:20,000; 60004-1-Ig; Proteintech), and anti-β-tubulin (1:5,000; Ab6046; Abcam).

Techniques: Immunofluorescence

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