Journal: Skeletal Muscle

Article Title: The breaking and making of healthy adult human skeletal muscle in vivo

doi: 10.1186/s13395-017-0142-x

Figure Lengend Snippet: Cross-sectional profiles of regenerating fibres. Three serial frozen cross sections of a biopsy obtained from regenerating vastus lateralis skeletal muscle 7 days after injury induced by electrical stimulation-elicited eccentric contractions (right). Three serial cross sections from the control (uninjured) leg of the same individual are shown (left) for comparison. The sections have been stained with alpha-sarcoglycan, beta-dystroglycan or dystrophin to label the sarcolemma, along with a basement membrane protein (laminin or collagen IV) and a myogenic marker (desmin or neonatal/embryonic myosin; MHCn/e). Each column of images contains single channel and combined images for each staining. In the injured muscle, dystrophin staining is completely absent in several fibres, while the basement membrane (laminin) is preserved. MHCn/e staining is evident in some small dystrophin-negative fibres. A similar pattern is evident from the alpha-sarcoglycan and beta-dystroglycan staining, with negative fibres, together with desmin+ cells, contained within a preserved basement membrane (collagen IV). Asterisk indicates some of the necrotic fibres. Note the different profiles of the injured fibres, such as varying fibre size, and infiltrating cells either confined to the fibre periphery or dispersed throughout the fibre. Scale bar, 100 μm

Article Snippet: Sections were stained with various combinations of antibodies against laminin, CD56, desmin and embryonic myosin (F1.652; Developmental Studies Hybridoma Bank); neonatal myosin (NCL-MHCn; Novocastra, Leica Microsystems A/S, Ballerup, Denmark); alpha-sarcoglycan (NCL-L-a-SARC, Novocastra); beta-dystroglycan (NCL-L-a-SARC, Novocastra); myogenin (F5d, Developmental Studies Hybridoma Bank); nestin, CD68, collagen IV and dystrophin (cat. no. D8168, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark); myosin type I (BA.D5, Developmental Studies Hybridoma Bank) and myosin type II (A4.74, Developmental Studies Hybridoma Bank).

Techniques: Control, Comparison, Staining, Membrane, Marker

Journal: Skeletal Muscle

Article Title: The breaking and making of healthy adult human skeletal muscle in vivo

doi: 10.1186/s13395-017-0142-x

Figure Lengend Snippet: Early sarcomere formation. Confocal images of regenerating zones of human single muscle fibres at 7 days post injury, where immature actin striations (phalloidin, red) were clearly visible within a desmin+ (green) filamentous sheath ( a ) or within a CD56+ membrane ( b ). The nuclei (blue) are likely to be myonuclei. Note part of the adjacent undamaged fibre at the bottom of the image in a , where the mature striation pattern of the sarcomere can be seen. The dashed line indicates the location of the YZ orthogonal view. Scale bars, 20 μm

Article Snippet: Sections were stained with various combinations of antibodies against laminin, CD56, desmin and embryonic myosin (F1.652; Developmental Studies Hybridoma Bank); neonatal myosin (NCL-MHCn; Novocastra, Leica Microsystems A/S, Ballerup, Denmark); alpha-sarcoglycan (NCL-L-a-SARC, Novocastra); beta-dystroglycan (NCL-L-a-SARC, Novocastra); myogenin (F5d, Developmental Studies Hybridoma Bank); nestin, CD68, collagen IV and dystrophin (cat. no. D8168, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark); myosin type I (BA.D5, Developmental Studies Hybridoma Bank) and myosin type II (A4.74, Developmental Studies Hybridoma Bank).

Techniques: Membrane

Journal: Skeletal Muscle

Article Title: The breaking and making of healthy adult human skeletal muscle in vivo

doi: 10.1186/s13395-017-0142-x

Figure Lengend Snippet: Myotube formation in vivo. a Single slice from a confocal stack of images of a regenerating zone of human skeletal muscle (day 7), capturing 6–7 adjoining nuclei (green) enclosed in a CD56+ (magenta) structure (arrow), likely a myotube, which is aligned along the fibre axis. Note the faintly visible adjoining uninjured fibre underneath, and part of another regenerating fibre at the bottom left of the image. The dashed line indicates the location of the YZ orthogonal view, where the fine CD56 staining can be seen entirely surrounding the myotube and where 2 peripherally located myonuclei are visible in the undamaged fibre. b Confocal images of 2 biopsy cross sections, from the same individual as in a , stained for CD56 and desmin, where CD56 can be seen encasing desmin+ structures, in 3 regenerating fibres (arrows) in each series of images. Scale bars, 20 μm

Article Snippet: Sections were stained with various combinations of antibodies against laminin, CD56, desmin and embryonic myosin (F1.652; Developmental Studies Hybridoma Bank); neonatal myosin (NCL-MHCn; Novocastra, Leica Microsystems A/S, Ballerup, Denmark); alpha-sarcoglycan (NCL-L-a-SARC, Novocastra); beta-dystroglycan (NCL-L-a-SARC, Novocastra); myogenin (F5d, Developmental Studies Hybridoma Bank); nestin, CD68, collagen IV and dystrophin (cat. no. D8168, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark); myosin type I (BA.D5, Developmental Studies Hybridoma Bank) and myosin type II (A4.74, Developmental Studies Hybridoma Bank).

Techniques: In Vivo, Staining

Journal: Skeletal Muscle

Article Title: The breaking and making of healthy adult human skeletal muscle in vivo

doi: 10.1186/s13395-017-0142-x

Figure Lengend Snippet: Myogenic and inflammatory cell activity viewed in serial cross sections. Microscope images of serial sections of one biopsy collected from regenerating human muscle 7 days after an injury protocol. Sixty serial sections (12 μm thick) were cut and stained for various markers. Shown here is a selection of these sections, where the section number (number sign) and proteins stained are indicated. Note the change in fibre diameter of the injured fibres, which are most clearly seen in section #19 (asterisk) by their lack of dystrophin (dys) staining and preserved basement membrane (collagen IV, col4). As observed in the single fibre analysis, these fibres are characterised by infiltration of macrophages (CD68+), the presence of neonatal/embryonic myosin (MHCne) and differentiating myogenic cells (desmin+, CD56+, myogenin+, nestin+). Scale bar 100 μm

Article Snippet: Sections were stained with various combinations of antibodies against laminin, CD56, desmin and embryonic myosin (F1.652; Developmental Studies Hybridoma Bank); neonatal myosin (NCL-MHCn; Novocastra, Leica Microsystems A/S, Ballerup, Denmark); alpha-sarcoglycan (NCL-L-a-SARC, Novocastra); beta-dystroglycan (NCL-L-a-SARC, Novocastra); myogenin (F5d, Developmental Studies Hybridoma Bank); nestin, CD68, collagen IV and dystrophin (cat. no. D8168, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark); myosin type I (BA.D5, Developmental Studies Hybridoma Bank) and myosin type II (A4.74, Developmental Studies Hybridoma Bank).

Techniques: Activity Assay, Microscopy, Staining, Selection, Membrane

Journal: Skeletal Muscle

Article Title: The breaking and making of healthy adult human skeletal muscle in vivo

doi: 10.1186/s13395-017-0142-x

Figure Lengend Snippet: Satellite cells on single fibres. Confocal images of satellite cells on the surface of regenerating human muscle fibres 30 days post injury. Three desmin+ (nestin-negative) satellite cells are visible in a , where nestin demonstrates a striated staining pattern of the myofibre, indicating ongoing regeneration. Scale bar 100 μm. Proliferating satellite cells are shown in b , evident from the expression of Ki67 and CD56, or desmin and nestin together with a nucleus undergoing mitosis. Scale bars, 20 μm

Article Snippet: Sections were stained with various combinations of antibodies against laminin, CD56, desmin and embryonic myosin (F1.652; Developmental Studies Hybridoma Bank); neonatal myosin (NCL-MHCn; Novocastra, Leica Microsystems A/S, Ballerup, Denmark); alpha-sarcoglycan (NCL-L-a-SARC, Novocastra); beta-dystroglycan (NCL-L-a-SARC, Novocastra); myogenin (F5d, Developmental Studies Hybridoma Bank); nestin, CD68, collagen IV and dystrophin (cat. no. D8168, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark); myosin type I (BA.D5, Developmental Studies Hybridoma Bank) and myosin type II (A4.74, Developmental Studies Hybridoma Bank).

Techniques: Staining, Expressing

Journal: Annals of neurology

Article Title: ASC1 is a cell cycle regulator associated with severe and mild forms of myopathy

doi: 10.1002/ana.25660

Figure Lengend Snippet: (A) Western blot of lysates from control (ctl) and three patient frozen muscles revealed altered expression of cyclins D1 and D3 as well as p21 in patients DII.2 (left panel), EII.4 and FIII.1 (right panel, control expression normalized to 1, dashed line). Absence of embryonic myosin (MHCe) in both patient DII.2 and control muscle samples excluded that this might be due to active muscle regeneration (including the presence of immature myofibers) in patients (lower panel, C2C12 used as a positive control). (B) Increased cyclin D1 and p21 expression in Trip4KD C2C12 with <20% ASC-1 residual expression (data not shown) compared to non-transfected cells (NT) or cells transfected with siRNA control (Scbl) (left and right panel, normalized to Scbl transfected cells; Mann–Whitney U, *p-value <0.05). (C) Rescue of the altered levels of cyclin D1 in Trip4KD C2C12 by wild-type human ASC-1. Western blot of protein extracts from Scbl (lanes 1 and 4), siTrip4 (lanes 2 and 5) or ctl C2C12 (lane 3) double-transfected with hASC-1 (lanes 3–5) 24h after endogenous ASC-1 silencing (upper panel). Quantification of the relative expression of cyclin D1 over tubulin (normalized for Scbl) in ctl, Scbl and siTrip4 cells in the presence or absence of hASC-1. A vector expressing hCelf1 was used as a non-specific control (lower panel). mASC-1= murine endogenous ASC-1; hASC-1 = human ectopic ASC-1; hCelf1 = human ectopic Celf1. (D) Western blot analysis of pRb phosphorylation state in NT, Scbl and siTrip4 cells revealed a fast migrating band corresponding to the hypo-phosphorylated form of Rb (pRb, 110 kDa) and slower migrating bands corresponding to hyper-phosphorylated forms of Rb (ppRb, 116kDa) (left panel). Red ponceau (redP) staining of the membrane showed similar loading in each condition. In all conditions, the hyper-phosphorylated forms of Rb were predominant compared to the hypo-phosphorylated form as expected for cycling cells. Although variability precluded reaching statistical significance, quantification of the ratio ppRb/total pRb for each condition (N=4) showed an increase by nearly 50% of hyper-phosphorylated forms in siTrip4 cells (right panel).

Article Snippet: Cell extracts (20 μg) were separated using 10% SDS-PAGE gel and transferred on nitrocellulose membranes (Bio-Rad Trans Turbo Transfer system) which were blocked and probed with the primary antibodies anti-ASC-1 (Abcam), anti-tubulin (Sigma), anti-p21 (Santa-Cruz), anti-cyclin D3 (Santa-Cruz), anti-cyclin D1 (Santa-Cruz or Millipore), anti-pRb (BD Pharmingen), anti-MHCe (DSHB) or anti-halotag (Promega).

Techniques: Western Blot, Control, Muscles, Expressing, Positive Control, Transfection, MANN-WHITNEY, Plasmid Preparation, Phospho-proteomics, Staining, Membrane