Journal: Molecular Medicine

Article Title: Adeno-associated Virus Serotype 8 (AAV8) Delivery of Recombinant A20 to Skeletal Muscle Reduces Pathological Activation of Nuclear Factor (NF)-?B in Muscle of mdx Mice

doi: 10.2119/molmed.2012.00299

Figure Lengend Snippet: Analysis of regeneration and necrosis in quadriceps of mdx mice. (A) Muscle sections of quadriceps from mdx mice treated with AAV8-tMCK-A20 (AAV8-A20) or saline were labeled with embryonic myosin heavy chain (eMyHC) antibody (red) or IgG (green) to assess the number of regenerating fibers and necrotic fibers, respectively. Quantification of the percentage of regenerating (B) and necrotic (C) fibers is shown. n = 9 for saline-treated mdx mice; n = 12 for AAV8-tMCK-A20–treated mdx mice. Scale bar = 100 μm; *p < 0.05.

Article Snippet: The monoclonal antibody to embryonic MHC (eMyHC) (F1.652), used to detect regenerating fibers, was obtained from the Developmental Studies Hybridoma Bank, developed by Helen Blau (University of Iowa, Department of Biological Sciences, Iowa City, IA, USA).

Techniques: Saline, Labeling

Journal: Journal of Anatomy

Article Title: Expression of MyHC‐15 and ‐ 2x in human muscle spindles: An immunohistochemical study

doi: 10.1111/joa.13923

Figure Lengend Snippet: Antibodies, their specificity and dilutions used to demonstrate MyHC isoforms in human intrafusal fibres.

Article Snippet: NCL MHCd , MyHC‐embryonic , 1:20 , Leica Biosystems Cat# NCL‐MHCd, RRID:AB_563901.

Techniques:

Journal: Journal of Anatomy

Article Title: Expression of MyHC‐15 and ‐ 2x in human muscle spindles: An immunohistochemical study

doi: 10.1111/joa.13923

Figure Lengend Snippet: Staining pattern of human intrafusal bag1, bag2 and chain fibres by antibodies against MyHC isoforms (slow‐tonic [st], 1, α, 2a, 2x, 2b, 15, embryonic [emb] and neonatal [neo]) in different regions (A, B, C) of muscle spindles in biceps brachii and flexor digitorum profundus muscles.

Article Snippet: NCL MHCd , MyHC‐embryonic , 1:20 , Leica Biosystems Cat# NCL‐MHCd, RRID:AB_563901.

Techniques: Staining, Muscles

Journal: EMBO Molecular Medicine

Article Title: Fibroadipogenic progenitors mediate the ability of HDAC inhibitors to promote regeneration in dystrophic muscles of young, but not old Mdx mice

doi: 10.1002/emmm.201202096

Figure Lengend Snippet: HDACi promote skeletal muscle regeneration only in actively regenerating muscles. A. Left panel: Right tibialis anterior of wild type mice were injured with notexin (+NTX) and the controlateral was left uninjured (−NTX). After 10 days of treatment with vehicle (Ctrl) or TSA, muscles were transverse sectioned and stained with an antibody against embryonal MyHC, (eMyHC – red) and nuclei counterstained with DAPI (blue). Right panel: Graph showing the % of eMyHC-positive myofibres. Data are represented as average ± SEM. n ≥ 3. Statistical significance assessed by t -test, ** p < 0.01. B. Left panel: immunofluorescence staining for eMyHC (red) and laminin (green) of tibialis anterior transverse sections of young (2 months old) and old (1 year old) mdx mice treated for 15 days with vehicle (Ctrl) or TSA. Nuclei were counterstained with DAPI (blue). Right panel: graph showing the % of eMyHC-positive myofibres. Data are represented as average ± SEM. n ≥ 3. Statistical significance assessed by t -test, * p < 0.05.

Article Snippet: Primary antibodies used were against: Laminin (Sigma; 1:100), embryonic MyHC (Developmental Studies Hybridoma Bank, DSHB; 1:20), MF-20 (DSHB, 1:20), MyoD (Santa Cruz, 1:20), GFP (Invitrogen, 1:500).

Techniques: Staining, Immunofluorescence

Journal: EMBO Molecular Medicine

Article Title: Fibroadipogenic progenitors mediate the ability of HDAC inhibitors to promote regeneration in dystrophic muscles of young, but not old Mdx mice

doi: 10.1002/emmm.201202096

Figure Lengend Snippet: Follistatin is soluble mediator of functional interactions between FAPs and MuSCs from mdx mice exposed to HDACi. A. FAPs and MuSCs were isolated from 1.5-month old mdx mice ctrl- and TSA-treated for 15 days. Cells were cultured for 7 days in growth medium (GM) and then Follistatin RNA levels were analysed by qRT-PCR. Follistatin is preferentially expressed by FAPs, as compared to MuSCs, and is strikingly up-regulated in FAPs isolated from TSA-treated mdx muscles. B. MuSCs from mdx mice and FAPs, from either mdx ctrl or TSA treated (15 days) mice, were co-cultured in transwell chambers (pore of 1 µm). FAPs were plated on the transwell insert and MuSCs were plated on the bottom compartment. After 7 days of co-cultures in the presence or absence of neutralizing antibodies against Follistatin, MuSCs were immunostained for MyHC (red) and nuclei counterstained by DAPI (blue). C. Graph showing the fusion index of the experiment represented in (B). We measured the percentage of nuclei that were MyHC − or MyHC + in mononucleated myotubes (<2), the % of nuclei that were inside myotubes containing between 2 and 5 nuclei (2 << 5) and the % of nuclei inside myotubes containing more than 5 nuclei (>5). Data are represented as average ± SEM, n ≥ 2. Statistical significance tested by one-way ANOVA, ** p < 0.01, *** p < 0.001. D. Follistatin RNAi in FAPs from ctrl mdx mice was performed by transfection of a shRNAi against follistatin (shFollistatin) and pSuper was used as ctrl. Graph shows the RNA levels of follistatin measured by qRT-PCR in FAPs upon RNAi. E. FAPs were treated (24 h) with TSA or not (ctrl) in vitro and subsequently co-cultured for 5 days in transwell, as described in B, with MuSCs isolated from mdx mice. MuSCs were then immunostained for MyHC (red) and nuclei counterstained by DAPI (blue). F. Graph showing the fusion index of the experiment represented in (E). We measured the percentage of nuclei that were MyHC − or MyHC + in mononucleated myotubes (<2), the % of nuclei that were inside myotubes containing between 2 and 5 nuclei (2<<5) and the % of nuclei inside myotubes containing more than 5 nuclei (>5). Data are represented as average ± SEM, n ≥ 2. Statistical significance tested by one-way ANOVA, * p < 0.05, ** p < 0.01.

Article Snippet: Primary antibodies used were against: Laminin (Sigma; 1:100), embryonic MyHC (Developmental Studies Hybridoma Bank, DSHB; 1:20), MF-20 (DSHB, 1:20), MyoD (Santa Cruz, 1:20), GFP (Invitrogen, 1:500).

Techniques: Functional Assay, Isolation, Cell Culture, Quantitative RT-PCR, Transfection, In Vitro

Journal: eLife

Article Title: Cellular localization of the cell cycle inhibitor Cdkn1c controls growth arrest of adult skeletal muscle stem cells

doi: 10.7554/eLife.33337

Figure Lengend Snippet: ( A ) Embryonic myosin (eMyHC)/LAMININ/DAPI, Hematoxylin and Eosin (HE), and Sirius red staining of twelve- to fifteen-week-old control (Ctrl) and Cdkn1c mutant mouse TA muscles were performed for histological and fibrosis characterization 4, 7 or thirty days after cardiotoxin (CTX) injection. Scale bars, 100 μm. ( B ) Fiber size (μm) distribution in control (Ctrl) and Cdkn1c mutant ( Cdkn1c -Mut) mice 7 (upper panel) or thirty (lower panel) days after CTX injection. ( C ) Histogram of average embryonic MyHC+ fiber diameters (μm) 4 days after CTX injection. ( D ) Histogram of average fiber diameters (μm) 7 and thirty days after CTX injection. ( E ) Fiber size (μm) distribution in control and Cdkn1c mutant mice thirty days after CTX injection. ( F ) Histogram of average fibrotic area per TA muscle. ( G ) PAX7+ (green) MuSCs (arrows) on the myofibers isolated from EDL muscles of Cdkn1c mutant and control mice thirty days after CTX injection. MYOD (red) is occasionally expressed in PAX7+ MuSCs (arrow heads). DAPI (blue) shows all nuclei. Scale bars, 50 μm. ( H ) Numbers of PAX7+ MuSCs on the EDL isolated myofibers . ( I ) Ratio of MYOD+ activated cells per PAX7+ MuSC on the myofibers isolated from EDL muscles of Cdkn1c mutant and control mice. Nuclei were counter-stained with DAPI. Scale bars, 100 μm. *p≤0.05, **p≤0.01.

Article Snippet: The following antibodies were used: chicken anti-GFP 1:1000 (#ab13970, Abcam), mouse anti-KI67 1:80 (#556003, BD Pharmingen), mouse anti-MYOD 1:80 (M3512, DAKO), rabbit anti-MYOD 1:1000 (sc304, Santa Cruz), mouse anti-MYOGENIN 1:100 (F5D-c, DSHB), mouse anti-embryonic MyHC 1:50 (F1.652, DSHB), mouse anti-embryonic MyHC 1:300 (sc53091, Santa Cruz), mouse anti-MyHC 1:100 (mf20-c, DSHB), rabbit AlexaFluor647-conjugated anti-laminin 1:200 (NB300-144AF647, Novus Biological), rabbit anti-laminin 1:400 (L9393, Sigma Aldrich), rat anti-laminin 1:1000 (4H8-2, Sigma-Aldrich), mouse anti-PAX7 1:100 (Pax7-c, DSHB), rabbit anti- CDKN1c 1:100 (sc8298, Santa Cruz), goat anti- CDKN1c 1:50 (sc1039, Santa Cruz), AlexaFluor-coupled secondary antibodies (Life Technologies, Jackson ImmunoResearch).

Techniques: Staining, Mutagenesis, Injection, Isolation

Journal: eLife

Article Title: Cellular localization of the cell cycle inhibitor Cdkn1c controls growth arrest of adult skeletal muscle stem cells

doi: 10.7554/eLife.33337

Figure Lengend Snippet: ( A ) Time-course of tamoxifen (TMX) administration, muscle satellite cell harvest (FACS arrow) and culture (light gray bar for growth culture conditions, dark gray bar for differentiation culture conditions). Analyzed animals were Pax7 CreERT2/+ ; Cdkn1c Flox(m)/+ ;Rosa mTmG ( Cdkn1c cKO) and Pax7 CreERT2/+ ; Cdkn1c +/+ ;Rosa mTmG (control; Ctrl); maternal inheritance of the imprinted Cdkn1c is indicated by superscript (m). ( B ) Cdkn1c transcript levels of control and Cdkn1c cKO myoblast cultures 3 days post-differentiation. ND; not detected. ( C–D ) Control ( C ) and Cdkn1c cKO ( D ) myoblast cultures were examined for CDKN1c protein (red) following three days under differentiation conditions. ( E–N ) Control and Cdkn1c cKO myoblast cultures were examined for EdU+ (light blue) cells ( E, G, H ), KI67+ cells ( F ), MYOGENIN+ cells (green; I, K, L ), and myotube formation ( J, M, N ). Nascent myotubes were marked with myosin heavy chain (MyHC; green; M, N ). Nuclei were counter-stained with DAPI (blue). Graphs show quantification of EdU and KI67 expression under growth conditions ( E, F ), MYOGENIN expression following 24 hr under differentiation conditions ( I ), and MyHC+ cells following 72 hr under differentiation conditions ( J ). Data show mean +SD, n = 3 animals. Asterisks indicate significance; *p≤0.05, ***p≤0.001. Scale bars, 40 μm ( C, K ), 1000 μm ( G, M ).

Article Snippet: The following antibodies were used: chicken anti-GFP 1:1000 (#ab13970, Abcam), mouse anti-KI67 1:80 (#556003, BD Pharmingen), mouse anti-MYOD 1:80 (M3512, DAKO), rabbit anti-MYOD 1:1000 (sc304, Santa Cruz), mouse anti-MYOGENIN 1:100 (F5D-c, DSHB), mouse anti-embryonic MyHC 1:50 (F1.652, DSHB), mouse anti-embryonic MyHC 1:300 (sc53091, Santa Cruz), mouse anti-MyHC 1:100 (mf20-c, DSHB), rabbit AlexaFluor647-conjugated anti-laminin 1:200 (NB300-144AF647, Novus Biological), rabbit anti-laminin 1:400 (L9393, Sigma Aldrich), rat anti-laminin 1:1000 (4H8-2, Sigma-Aldrich), mouse anti-PAX7 1:100 (Pax7-c, DSHB), rabbit anti- CDKN1c 1:100 (sc8298, Santa Cruz), goat anti- CDKN1c 1:50 (sc1039, Santa Cruz), AlexaFluor-coupled secondary antibodies (Life Technologies, Jackson ImmunoResearch).

Techniques: Staining, Expressing

Journal: eLife

Article Title: Cellular localization of the cell cycle inhibitor Cdkn1c controls growth arrest of adult skeletal muscle stem cells

doi: 10.7554/eLife.33337

Figure Lengend Snippet: ( A ) Control (Ctrl) and Cdkn1c mutant ( Cdkn1c -Mut) primary myoblasts are positive for both PAX7 and MYOD. ( B ) Under growth conditions, EdU+ cells are significantly higher in Cdkn1c mutant primary myoblasts compared with control cells. By contrast, there is no difference for MyHC+ differentiating cells. ( C ) Under differentiation conditions, Cdkn1c mutant primary myoblasts display reduced differentiation kinetics detected by MyHC in both day 1 and 3. However, in day 5, myogenic differentiation is almost saturated in both control and Cdkn1c mutant primary myoblasts. Nuclei were counter-stained with DAPI. Scale bars, 50 μm. *p≤0.05, **p≤0.01.

Article Snippet: The following antibodies were used: chicken anti-GFP 1:1000 (#ab13970, Abcam), mouse anti-KI67 1:80 (#556003, BD Pharmingen), mouse anti-MYOD 1:80 (M3512, DAKO), rabbit anti-MYOD 1:1000 (sc304, Santa Cruz), mouse anti-MYOGENIN 1:100 (F5D-c, DSHB), mouse anti-embryonic MyHC 1:50 (F1.652, DSHB), mouse anti-embryonic MyHC 1:300 (sc53091, Santa Cruz), mouse anti-MyHC 1:100 (mf20-c, DSHB), rabbit AlexaFluor647-conjugated anti-laminin 1:200 (NB300-144AF647, Novus Biological), rabbit anti-laminin 1:400 (L9393, Sigma Aldrich), rat anti-laminin 1:1000 (4H8-2, Sigma-Aldrich), mouse anti-PAX7 1:100 (Pax7-c, DSHB), rabbit anti- CDKN1c 1:100 (sc8298, Santa Cruz), goat anti- CDKN1c 1:50 (sc1039, Santa Cruz), AlexaFluor-coupled secondary antibodies (Life Technologies, Jackson ImmunoResearch).

Techniques: Mutagenesis, Staining

Journal: eLife

Article Title: Cellular localization of the cell cycle inhibitor Cdkn1c controls growth arrest of adult skeletal muscle stem cells

doi: 10.7554/eLife.33337

Figure Lengend Snippet: ( A ) Time-course of tamoxifen administration, intramuscular injury of TA muscle (CTX arrow), and muscle harvest (D7 arrow). ( B–G ) Cryosections of TA muscle were stained for histological and satellite cell population characterization 7 days after CTX injection. Analyzed animals at ( B-G ) were wild-type littermates (Wt; Pax7 + ; Cdkn1c + ; B–G ), Cre control ( Pax7 CreERT2 ; B’–G’ ), and Cdkn1c cKO ( Pax7 CreERT2 ; Cdkn1c Flox ; B’’–G’’ ). ( B ) HE staining for histologic characterization of the muscles. ( C ) Oil Red O staining for evaluation of fat infiltration of the muscles. ( D–E ) embryonic myosin (eMYHC, red)/LAMININ (LAM, green) immunofluorescence to mark newly formed myofibers post-regeneration. ( F–G ) PAX7 (red)/LAMININ (LAM, green) immunofluorescence to mark PAX7+ satellite cells. Nuclei in ( D-G ) were counter-stained with DAPI (blue). Scale bars, 50 μm. ( H ) Quantification of ( F-G ). Data show mean +SD, n ≥ 5 animals. Asterisks indicate significance; **p≤0.01.

Article Snippet: The following antibodies were used: chicken anti-GFP 1:1000 (#ab13970, Abcam), mouse anti-KI67 1:80 (#556003, BD Pharmingen), mouse anti-MYOD 1:80 (M3512, DAKO), rabbit anti-MYOD 1:1000 (sc304, Santa Cruz), mouse anti-MYOGENIN 1:100 (F5D-c, DSHB), mouse anti-embryonic MyHC 1:50 (F1.652, DSHB), mouse anti-embryonic MyHC 1:300 (sc53091, Santa Cruz), mouse anti-MyHC 1:100 (mf20-c, DSHB), rabbit AlexaFluor647-conjugated anti-laminin 1:200 (NB300-144AF647, Novus Biological), rabbit anti-laminin 1:400 (L9393, Sigma Aldrich), rat anti-laminin 1:1000 (4H8-2, Sigma-Aldrich), mouse anti-PAX7 1:100 (Pax7-c, DSHB), rabbit anti- CDKN1c 1:100 (sc8298, Santa Cruz), goat anti- CDKN1c 1:50 (sc1039, Santa Cruz), AlexaFluor-coupled secondary antibodies (Life Technologies, Jackson ImmunoResearch).

Techniques: Staining, Injection, Immunofluorescence

Journal: eLife

Article Title: Cellular localization of the cell cycle inhibitor Cdkn1c controls growth arrest of adult skeletal muscle stem cells

doi: 10.7554/eLife.33337

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: chicken anti-GFP 1:1000 (#ab13970, Abcam), mouse anti-KI67 1:80 (#556003, BD Pharmingen), mouse anti-MYOD 1:80 (M3512, DAKO), rabbit anti-MYOD 1:1000 (sc304, Santa Cruz), mouse anti-MYOGENIN 1:100 (F5D-c, DSHB), mouse anti-embryonic MyHC 1:50 (F1.652, DSHB), mouse anti-embryonic MyHC 1:300 (sc53091, Santa Cruz), mouse anti-MyHC 1:100 (mf20-c, DSHB), rabbit AlexaFluor647-conjugated anti-laminin 1:200 (NB300-144AF647, Novus Biological), rabbit anti-laminin 1:400 (L9393, Sigma Aldrich), rat anti-laminin 1:1000 (4H8-2, Sigma-Aldrich), mouse anti-PAX7 1:100 (Pax7-c, DSHB), rabbit anti- CDKN1c 1:100 (sc8298, Santa Cruz), goat anti- CDKN1c 1:50 (sc1039, Santa Cruz), AlexaFluor-coupled secondary antibodies (Life Technologies, Jackson ImmunoResearch).

Techniques: Generated, Plasmid Preparation, Affinity Purification, Sequencing, SYBR Green Assay, Modification, Software

Journal: Stem Cell Research & Therapy

Article Title: The role of the aging microenvironment on the fate of PDGFRβ lineage cells in skeletal muscle repair

doi: 10.1186/s13287-022-03072-y

Figure Lengend Snippet: Muscle regeneration capacity of PDGFRβ lineage cells decrease during aging. A , B Images representing GFP + cells centrally nucleated muscle fibers in young and old muscles 5 days after CTX injury. The images in ( B ) are higher magnification of the images in ( A ). C Quantification of regenerated GFP + muscle fibers in skeletal muscle of young and old mice after injury. D Images representing GFP and eMyHC staining. E Quantification of eMyHC + muscle fibers. F Quantification of both GFP + and eMyHC + muscle fibers. N = 6, error bars indicate ‘mean + SD’, * p < 0.05. In panel ( A ), all scale bars = 200 µm; in panel ( B ) and ( D ), all scale bars = 50 µm

Article Snippet: A Mouse on Mouse kit (BMK-2202, Vector, Olean, NY, USA) was used for Pax7 (DSHB, 1:100) and eMyHC (DSHB, F1.652C,1:50) staining according to the manufacturer’s protocol.

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: APC is required for muscle stem cell proliferation and skeletal muscle tissue repair

doi: 10.1083/jcb.201501053

Figure Lengend Snippet: APC is required for adult skeletal muscle regeneration. (A) TM regimen and cardiotoxin (CTX) injury scheme for control and APC SC-KO mice. (B and C) Immunostaining for Pax7 (B) and e-MyHC (C) on TA cryosections 4 d after injury. (D–L) Analysis of TA muscles 14 d after injury reveals that tissue regeneration is defective in APC SC-KO muscles. (D) Whole sections of TA muscles immunostained for Laminin. Immunostaining for Tcf4 (F), type 1 Collagen (H), and Pax7 and Laminin (L) on TA cryosections is shown. Quantification of TA weight 14 d after injury normalized by uninjured TA weight (E), of the number of Tcf4+ cells/mm 2 (G), of relative Collagen+ area (I), and of sublaminar Pax7+ cells (normalized by Pax7+ cells of uninjured TA; J) is also shown. (K) GFP immunostaining on transverse sections of TAs isolated from Pax7 CreER ;Z/EG (Control-Z/EG) and Pax7 CreER APC lox/lox ;Z/EG (APC SC-KO-Z/EG) 14 d after injury demonstrates that APC-null satellite cells do not give rise to new myofibers, nor trans-differentiate in other cell types. Bars: (B, C, F, H, K, and L) 50 µm; (D) 200 µm. Nuclei are stained with Hoechst. Error bars indicate SEM. Inset panels show a 2× enlargement.

Article Snippet: Primary antibodies were as follows: mouse β-catenin (BD), goat Collagen Type I (SouthernBiotech), rabbit GFP (Life Technologies), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), mouse MyoD (Dako), mouse embryonic-MyHC (Developmental Studies Hybridoma Bank), mouse Pax7 (DSHB), and rabbit Tcf4 (Cell Signaling Technology).

Techniques: Immunostaining, Isolation, Staining