mouse anti-myod Search Results


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  • 96
    Millipore monoclonal anti myod1 antibody
    HIF2A stabilization under normoxia promotes quiescence, self-renewal, and stemness of SCs yet impedes myogenic differentiation. ( A ) Diagram depicting the timeline and plasmids used for HIF2A stabilization in normoxic SC culture. Pound signs denote the number and locations of point mutations in HIF2ATM. ( B ) Representative images of transfected (GFP + ) SC clusters on myofibers from C57BL/6 mice ( n > 50 myofibers from 7 mice/group). The SC clusters were transfected with either HIF2ATM or control plasmids and stained for Pax7, <t>MyoD,</t> and DAPI. Scale bar: 5 μm. ( C ) Number of SC clusters, Pax7 + SCs per SC cluster, and Pax7 + MyoD – , Pax7 + MyoD + , and Pax7 – MyoD + SCs per SC cluster ( n > 50 myofibers). ( D ) Diagram of the experimental scheme for transplantation of HIF2A-stabilized SCs and tracing of their cell fates in vivo. ( E ) Cross-sectional images of TA muscles that were transplanted with HIF2ATM- or control plasmid–transfected SCs ( n = 5 mice/group; 21 days after the first injury). Immunofluorescence of Pax7 and nmGFP revealed 2 fates of transplanted SCs: engrafted SCs that retained stemness (nmGFP + Pax7 + ; circles, bottom) and engrafted SCs that differentiated into myonuclei (nmGFP + Pax7 – ; arrowheads). Scale bars: 20 μm. ( F and H ) Percentage of engrafted and self-renewed nmGFP + Pax7 + SCs in the total SC pool after the first round (21 dpi; n = 5 mice/group in F ) and second round (30 dpi; n = 6 mice/group in H ) of regeneration. ( G and I ) Number of nmGFP + Pax7 – differentiated myonuclei per TA muscle section after the first round ( G ) and second round ( I ) of regeneration. * P
    Monoclonal Anti Myod1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse anti myod1
    Stat3 promotes myogenic lineage progression in cultured satellite cells. ( a ) Left, representative images of satellite cells isolated by FACS, cultured in vitro and analyzed 5 d after isolation (green, pSTAT3; red, <t>Myod1;</t> blue, nuclei). Scale bar, 50 µm.
    Mouse Anti Myod1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies mouse anti myod1
    IL34 is required for myogenic lineage progression in cultured SCs. a Fresh SCs were seeded, cultured in growth medium, and then induced to differentiate for various amounts of time. The immunoblots presented here reveal the protein levels of IL34, p-STAT3, STAT3, and unrelated β-tubulin in cultured SCs at different time points after initiation of differentiation. b SCs were isolated from the hindlimbs of WT mice using FACS, cultured in growth medium for 3 days, and treated with shIL34 lentivirus or control shRNA (shScr) for 36 h. The positively infected SCs were then purified by FACS according to the intrinsic enhanced green fluorescent protein (EGFP) fluorescence expressed by the lentivirus. Purified infected SCs were cultured for further analysis. c Western blot analysis of the levels of IL34, p-STAT3, STAT3, and an unrelated protein (GAPDH) in WT cell cultures after shScr and shIL34 treatment. d Western blot analysis of the levels of MyHC, <t>MyoD,</t> Pax7, and an unrelated protein (GAPDH) in WT cell cultures after shScr and shIL34 treatment. e Representative merged photomicrographs of SCs stably infected with a lentivirus expressing shIL34 or shScr that were induced to differentiate for one day and stained for Pax7 and DAPI. Scale bar: 30 μm. f Quantification of the percentage of Pax7 + SCs after shScr and shIL34 infection. *** P
    Mouse Anti Myod1, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology mouse anti myod1
    Successful myotube differentiation of 3-deazaneplanocin A hydrochloride (DZNep)-treated <t>MYOD1</t> -urine-derived cells (UDCs) derived from healthy individuals. ( A ) Representative images of immunocytochemistry for MyHC (upper) and DYSTROPHIN (lower) in MYOD1 -UDCs from healthy children and adults 14 days after differentiation. HI: healthy individual. #1: 8-year-old male; #2: 13-year-old male; #3: 33-year-old male; #4: 38-year-old male. Scale bar: 100 μm. ( B ) Comparison of fusion index with and without DZNep treatment. The fusion index was calculated as a percentage of nuclei inside the MyHC positive myotubes using randomly selected three pictures from four healthy individuals respectively. The Mann-Whitney test was used for comparisons; ***P
    Mouse Anti Myod1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology rabbit anti mouse myod
    Marker expression in SC clones cultured under normoxia and hypoxia. Immunofluorescence analyses of LPC and HPC cultured for 5 days at 20% and 2% O 2 revealed differences in the expression of <t>MyoD,</t> <t>Myf5</t> (only at 2% O 2 ) and CD34. Phase-contrast and corresponding immunostaining examples for the specific marker (in red) merged with DAPI (left, scale bar = 100 µm, insets with higher magnification). Graphs indicate the percentage of marker-positive cells per clone (right, mean±SEM, * p
    Rabbit Anti Mouse Myod, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Abcam mouse anti myod1
    Myogenic marker expression of isolated SC/MPC populations. Flow cytometric analysis of immunofluorescence-stained SC/MPC from SM and LD muscle cultured for 4 days ( a ) and 8 days ( b ) in growth medium. The cells were immunostained for Pax7, <t>MyoD1,</t> MyoG, and Desmin. Percentages of positive cells in each sample are presented as Box-Whisker plots with the median and the maximum 1.5 of the interquartile range (Q1-Q3). Outliers are included in the figure as circles but were excluded from statistical analysis. All populations expressed the selected myogenic markers, whereas a higher proportion of cells were positive for MyoG and Desmin, which are markers for terminally committed myogenic cells. Statistical analysis was performed by ANOVA (with the Holm-Sidak method as a pairwise multiple comparison procedure) or the Kruskal-Wallis One-Way ANOVA on Ranks (with Dunn’s method as a pairwise multiple comparison procedure), *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, n = 4–9 animals
    Mouse Anti Myod1, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson mouse monoclonal anti myod
    Schematic of C2C12 Differentiation (A) In the C2C12 cell culture system, upon removal of serum, proliferating myoblasts can de-differentiate into quiescent reserve cells. Reserve cells are capable of differentiating into myoblasts with the addition of serum. Myoblasts are <t>Pax7</t> + <t>/MyoD</t> + ; whereas, reserve cells are Pax7 + /MyoD − . Cells are guided to the reserve cell fate by expression and activation of the Notch receptor, which gives elevated Hes and Hey gene expression and maintains the self-renewing state. (B) Upon serum removal, C2C12 cells also differentiate into myocytes, which are Pax7/MyoD + . Myocytes lose their proliferative capabilities, express myogenin (MyoG), followed by myosin heavy chain (MHC), and ultimately fuse to form syncytial myotubes.
    Mouse Monoclonal Anti Myod, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies mouse monoclonal anti myod
    Myogenic progression in S1PR3 -null satellite cells is relatively normal. EDL myofibres and their associated satellite cells were isolated from either age-matched wild-type ( S1PR3 +/+ ) or S1PR3- null ( S1PR3 −/− ) mice and cultured in proliferation medium for 72 h. (A)–(C) Samples were taken at 24 h intervals, fixed and co-immunostained for <t>Pax7,</t> <t>MyoD</t> and Myogenin, which showed a general increase in each population when S1PR3 was absent, consistent with enhanced proliferation. (D) When expressed as a ratio though, the proportions of cells expressing Pax7, MyoD or myogenin were similar between wildtype and S1PR3 -null mice. (E) and (F) Expanded satellite cell-derived myoblasts were also plated at high confluency and then cultured in differentiation medium for 48 h before immunostaining for Myosin Heavy Chain (MyHC) and counterstaining with DAPI. Counting the number of nuclei within MyHC+ve myotubes ( > 2 nuclei) to determine the fusion index revealed that more satellite cells from S1PR3 -null mice had differentiated and fused than from control mice ((E), quantified (F)). Values are expressed as mean±SEM from multiple samples from at least three mice ( n =3/4) where an asterisk denotes significantly different ( p
    Mouse Monoclonal Anti Myod, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology mouse monoclonal anti myod antibody
    Changes in the markers of satellite cells . Representative cross-sections of soleus muscle immunostained for M-cadherin, <t>BrdU,</t> <t>myoD,</t> and myogenin (green, arrows ), respectively. The two columns on the left are DIC images. The two columns on the right show dystrophin immunolabeling (red) to identify fiber profiles and nuclear staining with DAPI (blue). Scale bar = 20 μm.
    Mouse Monoclonal Anti Myod Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Novocastra mouse anti myod
    Myogenesis in adherent colonies derived from fiber-associated satellite cells. Satellite cell colonies were stained for c-met (red) and <t>MyoD</t> (green; A,C,E ) or marker myosin heavy chain (red; B,D,F ); nuclei were visualized with DAPI. Wild-type colonies form multinucleate myotubes, stain positively for MyoD ( A ), and express myosin heavy chain, indicating successful differentiation ( B ). <t>Syndecan-3</t> -/- colonies form large, irregular syncytia, rarely express MyoD ( C ), and fail to express myosin heavy chain ( D ). Syndecan-4 -/- colonies completely fail to either form myotubes or to express MyoD ( E ) and only very rarely do single cells express myosin heavy chain ( F ). ( G ) Addition of exogenous heparin increased the number of cells per clone in mass cultures of wild-type and syndecan-3 -/- satellite cells but not syndecan-4 -/- satellite cells; bars represent standard deviations. Heparin also increased differentiation as measured by expression of nuclear MyoD (red) and myosin heavy chain (green) in syndecan-3 -/- cells (525 μg/mL shown; H ) and partially rescued the mutant phenotype in syndecan-4 -/- cells (525 μg/mL shown; I ).
    Mouse Anti Myod, supplied by Novocastra, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam anti myod1 mouse monoclonal
    Histological changes on day five after empty vector or BMP-2 gene transfer. a-e) Skeletal muscles on day five after empty vector transfer. a) Haematoxylin-eosin staining showing spindle-shaped and cytoplasm-enriched cells in the spaces between muscle fibres (arrows). b) CD68-positive cells (arrow). c) Pax7-positive cells (arrow). d) <t>Myod1-</t> positive cells (arrow). e) Myogenin-positive cells are detected around the cell migration area (arrows). f-k) Skeletal muscles on day five after BMP-2 gene transfer. f ) Haematoxylin-eosin staining showing typical muscle fibres. g) CD68-positive cells. h) BMP-2-positive cells are detected in the haematoxylin-positive population, but not in muscle fibres. i) Pax7-positive cells (arrow). j) M-cadherin-positive cells (arrows). k) Myogenin-positive cells (arrows) are detected between muscle fibres. Scale bars: a,b) 100 > m; c-k) 50 > m.
    Anti Myod1 Mouse Monoclonal, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson mouse anti human myod
    dKO Cells Exhibit a Block in Muscle Differentiation (A–L) Expression of <t>myogenin</t> (MYOG) in injured muscle is dependent on <t>MyoD</t> or Myf5 . MYOG expression (arrowheads) was delayed or undetectable in Myf5-SA (F and H) and dKO (J and L) satellite cells, respectively. (M–T) Cultured dKO satellite cells fail to fuse or express MYOG or MyHC after 21 days in DM. (U–X) Co-culturing of equivalent numbers of wild-type and GFP+ dKO cells for 14 days in DM did not rescue fusion or differentiation capacity of dKO cells. Arrowheads identify representative fibers, which are GFP– and MyHC+. Scale bars represent 10 μm (A–L), 50 μm (M–T), or 100 μm (U–X).
    Mouse Anti Human Myod, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen mouse anti myod antibody 5a8
    dKO Cells Exhibit a Block in Muscle Differentiation (A–L) Expression of <t>myogenin</t> (MYOG) in injured muscle is dependent on <t>MyoD</t> or Myf5 . MYOG expression (arrowheads) was delayed or undetectable in Myf5-SA (F and H) and dKO (J and L) satellite cells, respectively. (M–T) Cultured dKO satellite cells fail to fuse or express MYOG or MyHC after 21 days in DM. (U–X) Co-culturing of equivalent numbers of wild-type and GFP+ dKO cells for 14 days in DM did not rescue fusion or differentiation capacity of dKO cells. Arrowheads identify representative fibers, which are GFP– and MyHC+. Scale bars represent 10 μm (A–L), 50 μm (M–T), or 100 μm (U–X).
    Mouse Anti Myod Antibody 5a8, supplied by Pharmingen, used in various techniques. Bioz Stars score: 80/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher myod alexa fluor 488 conjugated affinipure goat anti mouse igg h l
    dKO Cells Exhibit a Block in Muscle Differentiation (A–L) Expression of <t>myogenin</t> (MYOG) in injured muscle is dependent on <t>MyoD</t> or Myf5 . MYOG expression (arrowheads) was delayed or undetectable in Myf5-SA (F and H) and dKO (J and L) satellite cells, respectively. (M–T) Cultured dKO satellite cells fail to fuse or express MYOG or MyHC after 21 days in DM. (U–X) Co-culturing of equivalent numbers of wild-type and GFP+ dKO cells for 14 days in DM did not rescue fusion or differentiation capacity of dKO cells. Arrowheads identify representative fibers, which are GFP– and MyHC+. Scale bars represent 10 μm (A–L), 50 μm (M–T), or 100 μm (U–X).
    Myod Alexa Fluor 488 Conjugated Affinipure Goat Anti Mouse Igg H L, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    HIF2A stabilization under normoxia promotes quiescence, self-renewal, and stemness of SCs yet impedes myogenic differentiation. ( A ) Diagram depicting the timeline and plasmids used for HIF2A stabilization in normoxic SC culture. Pound signs denote the number and locations of point mutations in HIF2ATM. ( B ) Representative images of transfected (GFP + ) SC clusters on myofibers from C57BL/6 mice ( n > 50 myofibers from 7 mice/group). The SC clusters were transfected with either HIF2ATM or control plasmids and stained for Pax7, MyoD, and DAPI. Scale bar: 5 μm. ( C ) Number of SC clusters, Pax7 + SCs per SC cluster, and Pax7 + MyoD – , Pax7 + MyoD + , and Pax7 – MyoD + SCs per SC cluster ( n > 50 myofibers). ( D ) Diagram of the experimental scheme for transplantation of HIF2A-stabilized SCs and tracing of their cell fates in vivo. ( E ) Cross-sectional images of TA muscles that were transplanted with HIF2ATM- or control plasmid–transfected SCs ( n = 5 mice/group; 21 days after the first injury). Immunofluorescence of Pax7 and nmGFP revealed 2 fates of transplanted SCs: engrafted SCs that retained stemness (nmGFP + Pax7 + ; circles, bottom) and engrafted SCs that differentiated into myonuclei (nmGFP + Pax7 – ; arrowheads). Scale bars: 20 μm. ( F and H ) Percentage of engrafted and self-renewed nmGFP + Pax7 + SCs in the total SC pool after the first round (21 dpi; n = 5 mice/group in F ) and second round (30 dpi; n = 6 mice/group in H ) of regeneration. ( G and I ) Number of nmGFP + Pax7 – differentiated myonuclei per TA muscle section after the first round ( G ) and second round ( I ) of regeneration. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Transient HIF2A inhibition promotes satellite cell proliferation and muscle regeneration

    doi: 10.1172/JCI96208

    Figure Lengend Snippet: HIF2A stabilization under normoxia promotes quiescence, self-renewal, and stemness of SCs yet impedes myogenic differentiation. ( A ) Diagram depicting the timeline and plasmids used for HIF2A stabilization in normoxic SC culture. Pound signs denote the number and locations of point mutations in HIF2ATM. ( B ) Representative images of transfected (GFP + ) SC clusters on myofibers from C57BL/6 mice ( n > 50 myofibers from 7 mice/group). The SC clusters were transfected with either HIF2ATM or control plasmids and stained for Pax7, MyoD, and DAPI. Scale bar: 5 μm. ( C ) Number of SC clusters, Pax7 + SCs per SC cluster, and Pax7 + MyoD – , Pax7 + MyoD + , and Pax7 – MyoD + SCs per SC cluster ( n > 50 myofibers). ( D ) Diagram of the experimental scheme for transplantation of HIF2A-stabilized SCs and tracing of their cell fates in vivo. ( E ) Cross-sectional images of TA muscles that were transplanted with HIF2ATM- or control plasmid–transfected SCs ( n = 5 mice/group; 21 days after the first injury). Immunofluorescence of Pax7 and nmGFP revealed 2 fates of transplanted SCs: engrafted SCs that retained stemness (nmGFP + Pax7 + ; circles, bottom) and engrafted SCs that differentiated into myonuclei (nmGFP + Pax7 – ; arrowheads). Scale bars: 20 μm. ( F and H ) Percentage of engrafted and self-renewed nmGFP + Pax7 + SCs in the total SC pool after the first round (21 dpi; n = 5 mice/group in F ) and second round (30 dpi; n = 6 mice/group in H ) of regeneration. ( G and I ) Number of nmGFP + Pax7 – differentiated myonuclei per TA muscle section after the first round ( G ) and second round ( I ) of regeneration. * P

    Article Snippet: For staining of myofibers and myoblasts, the myofibers and myoblasts were fixed in PFA (4%, 10 min), blocked in 5% BSA/5% normal goat serum/PBS, and incubated with the following primary antibodies: anti-Pax7 (1:50); anti-MyoD (1:250; MilliporeSigma; M6190); anti-HIF2A (1:250); anti-HIF1A (1:250); and anti-GFP (1:1,000; 101Bio; P601).

    Techniques: Transfection, Mouse Assay, Staining, Transplantation Assay, In Vivo, Plasmid Preparation, Immunofluorescence

    Transient pharmacological inhibition of HIF2A in CTX-injured muscle promotes SC proliferation and accelerates muscle regeneration. ( A ) Timeline of pharmacological inhibition of HIF2A after CTX-induced muscle injury in C57BL/6 mice. ( B ) Representative images of HIF-c2– or 1% DMSO–treated TA muscles (3 dpi) from C57BL/6 mice ( n = 6 mice/group). Immunofluorescence revealed increased Pax7 + EdU + proliferative SCs (arrowheads) and decreased Pax7 + EdU – QSCs (circles). Scale bar: 20 μm. ( C ) Number of EdU + and EdU – SCs per TA muscle section 3 dpi ( n = 6). ( D ) Number of Pax7 + SCs per TA muscle section 7 dpi, 10 dpi, and 30 dpi ( n = 6 or 7). ( E ) Representative eMyHC and laminin B2 immunofluorescence in HIF-c2– or DMSO-treated TA muscles 7 dpi and 30 dpi ( n = 3). Scale bars: 20 μm. ( F ) Immunoblots showing the expression levels of HIF2A, eMyHC, MyoD, and tubulin in HIF-c2– or DMSO-treated TA muscles 7 dpi ( n = 3). ( G ) Distributions of myofiber cross-sectional areas of HIF-c2– or DMSO-treated TA muscles 30 dpi ( n = 3). ( H ) Maximal torques of uninjured TA muscles and HIF-c2– or DMSO-treated TA muscles 7 dpi ( n = 6), 10 dpi ( n = 3), and 30 dpi ( n = 3). ( I ) Timeline of pharmacological inhibition of HIF2A after CTX-induced muscle injury in C57BL/6 mice and SC-HIF2AKO mice. ( J )Representative Pax7 immunofluorescence images of HIF-c2– or DMSO-treated TA muscles from SC-HIF2AKO mice ( n = 6 mice/group) 10 dpi and number of Pax7 + SCs per TA muscle section 7 dpi. Scale bar: 20 um. ( K ) Representative eMyHC and laminin B2 immunofluorescence images of HIF-c2– or DMSO-treated TA muscles from SC-HIF2AKO ( n = 6 mice/group) 7 dpi. Scale bar: 20 μm. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Transient HIF2A inhibition promotes satellite cell proliferation and muscle regeneration

    doi: 10.1172/JCI96208

    Figure Lengend Snippet: Transient pharmacological inhibition of HIF2A in CTX-injured muscle promotes SC proliferation and accelerates muscle regeneration. ( A ) Timeline of pharmacological inhibition of HIF2A after CTX-induced muscle injury in C57BL/6 mice. ( B ) Representative images of HIF-c2– or 1% DMSO–treated TA muscles (3 dpi) from C57BL/6 mice ( n = 6 mice/group). Immunofluorescence revealed increased Pax7 + EdU + proliferative SCs (arrowheads) and decreased Pax7 + EdU – QSCs (circles). Scale bar: 20 μm. ( C ) Number of EdU + and EdU – SCs per TA muscle section 3 dpi ( n = 6). ( D ) Number of Pax7 + SCs per TA muscle section 7 dpi, 10 dpi, and 30 dpi ( n = 6 or 7). ( E ) Representative eMyHC and laminin B2 immunofluorescence in HIF-c2– or DMSO-treated TA muscles 7 dpi and 30 dpi ( n = 3). Scale bars: 20 μm. ( F ) Immunoblots showing the expression levels of HIF2A, eMyHC, MyoD, and tubulin in HIF-c2– or DMSO-treated TA muscles 7 dpi ( n = 3). ( G ) Distributions of myofiber cross-sectional areas of HIF-c2– or DMSO-treated TA muscles 30 dpi ( n = 3). ( H ) Maximal torques of uninjured TA muscles and HIF-c2– or DMSO-treated TA muscles 7 dpi ( n = 6), 10 dpi ( n = 3), and 30 dpi ( n = 3). ( I ) Timeline of pharmacological inhibition of HIF2A after CTX-induced muscle injury in C57BL/6 mice and SC-HIF2AKO mice. ( J )Representative Pax7 immunofluorescence images of HIF-c2– or DMSO-treated TA muscles from SC-HIF2AKO mice ( n = 6 mice/group) 10 dpi and number of Pax7 + SCs per TA muscle section 7 dpi. Scale bar: 20 um. ( K ) Representative eMyHC and laminin B2 immunofluorescence images of HIF-c2– or DMSO-treated TA muscles from SC-HIF2AKO ( n = 6 mice/group) 7 dpi. Scale bar: 20 μm. * P

    Article Snippet: For staining of myofibers and myoblasts, the myofibers and myoblasts were fixed in PFA (4%, 10 min), blocked in 5% BSA/5% normal goat serum/PBS, and incubated with the following primary antibodies: anti-Pax7 (1:50); anti-MyoD (1:250; MilliporeSigma; M6190); anti-HIF2A (1:250); anti-HIF1A (1:250); and anti-GFP (1:1,000; 101Bio; P601).

    Techniques: Inhibition, Mouse Assay, Immunofluorescence, Western Blot, Expressing

    Genetic ablation of HIF2A in QSCs leads to transient activation, proliferation, and differentiation of SCs. ( A ) Timeline of genetic ablation of HIF2A in QSCs. ( B ) Representative images of myofibers from SC-HIF2AKO mice and control littermates ( n > 50 myofibers from 5 mice/group; 10 dpr). Immunofluorescence of Pax7 (red), HIF2A (green), MyoD (purple), and DAPI (blue) staining revealed HIF2A – MyoD + and HIF2A + MyoD – SCs (arrowheads) in SC-HIF2AKO and control mice, respectively. Scale bar: 10 μm. ( C ) Number of HIF2A + and HIF2A – SCs per myofiber (10 dpr). ( D ) Number of MyoD – and MyoD + SCs per myofiber (10 dpr). ( E ) Timeline characterizing SC proliferation after HIF2A ablation in QSCs. ( F ) Representative cross-sectional images of TA muscles from SC-HIF2AKO mice and control littermates ( n = 6 mice/group; 16 dpr). Immunofluorescence of Pax7 (red), Ki67 (green), EdU (purple), and DAPI (blue) staining revealed an increase in Ki67 + EdU + SCs (arrowheads) in SC-HIF2AKO mice. Scale bar: 20 μm. ( G ) Number of Ki67 – and Ki67 + SCs per TA section. ( H ) Number of EdU – and EdU + SCs per TA section. ( I ) Timeline for tracing SC fates after HIF2A ablation in QSCs. ( J ) Representative images of TA muscles from SC-HIF2AKO-INTACT and control SC-INTACT mice ( n = 6 mice/group; 16 dpr). Immunofluorescence of nmGFP, Pax7, laminin B2, and DAPI revealed increased nmGFP + Pax7 + SCs (arrowheads) and nmGFP + Pax7 – myonuclei (asterisks) in SC-HIF2AKO-INTACT mice. Scale bar: 20 μm and 5 μm (insets). Inset images show that both nmGFP + Pax7 + SCs and nmGFP + Pax7 – myonuclei are adjacent to the basal lamina. ( K ) Number of nmGFP + Pax7 + SCs and nmGFP + Pax7 – myonuclei per TA section. ( L ) Number of nmGFP + myogenin + differentiating SCs per EDL myofiber (16 dpr). ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Transient HIF2A inhibition promotes satellite cell proliferation and muscle regeneration

    doi: 10.1172/JCI96208

    Figure Lengend Snippet: Genetic ablation of HIF2A in QSCs leads to transient activation, proliferation, and differentiation of SCs. ( A ) Timeline of genetic ablation of HIF2A in QSCs. ( B ) Representative images of myofibers from SC-HIF2AKO mice and control littermates ( n > 50 myofibers from 5 mice/group; 10 dpr). Immunofluorescence of Pax7 (red), HIF2A (green), MyoD (purple), and DAPI (blue) staining revealed HIF2A – MyoD + and HIF2A + MyoD – SCs (arrowheads) in SC-HIF2AKO and control mice, respectively. Scale bar: 10 μm. ( C ) Number of HIF2A + and HIF2A – SCs per myofiber (10 dpr). ( D ) Number of MyoD – and MyoD + SCs per myofiber (10 dpr). ( E ) Timeline characterizing SC proliferation after HIF2A ablation in QSCs. ( F ) Representative cross-sectional images of TA muscles from SC-HIF2AKO mice and control littermates ( n = 6 mice/group; 16 dpr). Immunofluorescence of Pax7 (red), Ki67 (green), EdU (purple), and DAPI (blue) staining revealed an increase in Ki67 + EdU + SCs (arrowheads) in SC-HIF2AKO mice. Scale bar: 20 μm. ( G ) Number of Ki67 – and Ki67 + SCs per TA section. ( H ) Number of EdU – and EdU + SCs per TA section. ( I ) Timeline for tracing SC fates after HIF2A ablation in QSCs. ( J ) Representative images of TA muscles from SC-HIF2AKO-INTACT and control SC-INTACT mice ( n = 6 mice/group; 16 dpr). Immunofluorescence of nmGFP, Pax7, laminin B2, and DAPI revealed increased nmGFP + Pax7 + SCs (arrowheads) and nmGFP + Pax7 – myonuclei (asterisks) in SC-HIF2AKO-INTACT mice. Scale bar: 20 μm and 5 μm (insets). Inset images show that both nmGFP + Pax7 + SCs and nmGFP + Pax7 – myonuclei are adjacent to the basal lamina. ( K ) Number of nmGFP + Pax7 + SCs and nmGFP + Pax7 – myonuclei per TA section. ( L ) Number of nmGFP + myogenin + differentiating SCs per EDL myofiber (16 dpr). ** P

    Article Snippet: For staining of myofibers and myoblasts, the myofibers and myoblasts were fixed in PFA (4%, 10 min), blocked in 5% BSA/5% normal goat serum/PBS, and incubated with the following primary antibodies: anti-Pax7 (1:50); anti-MyoD (1:250; MilliporeSigma; M6190); anti-HIF2A (1:250); anti-HIF1A (1:250); and anti-GFP (1:1,000; 101Bio; P601).

    Techniques: Activation Assay, Mouse Assay, Immunofluorescence, Staining

    Expression levels of MyoD1 , Myog , and Myh2 mRNA in bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs). * P

    Journal: Anatomy & Cell Biology

    Article Title: Expression of surface markers and myogenic potential of rat bone marrow- and adipose-derived stem cells: a comparative study

    doi: 10.5115/acb.2013.46.2.113

    Figure Lengend Snippet: Expression levels of MyoD1 , Myog , and Myh2 mRNA in bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs). * P

    Article Snippet: Next, cells were blocked in 10% normal goat serum (Sigma-Aldrich) and incubated with mouse monoclonal anti-MyoD1 (1:100), mouse monoclonal anti-myogenin (1:100), or mouse monoclonal anti-myosin (fast skeletal, 1:100, Sigma-Aldrich) overnight at 4℃.

    Techniques: Expressing, Derivative Assay

    Expression and the intracellular position of myogenic markers MyoD1, Myog, and myosin (fast skeletal) in bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs) in the absence or presence of chemical growth factors. MyoD1 (A) and Myog (B), as skeletal muscle transcription factors, were mostly concentrated in the nuclei (arrows). (C) Myosin (fast skeletal). Undifferentiated stem cells were taken as a negative control, and the cell line L6 was considered as positive control (A-C, ×630).

    Journal: Anatomy & Cell Biology

    Article Title: Expression of surface markers and myogenic potential of rat bone marrow- and adipose-derived stem cells: a comparative study

    doi: 10.5115/acb.2013.46.2.113

    Figure Lengend Snippet: Expression and the intracellular position of myogenic markers MyoD1, Myog, and myosin (fast skeletal) in bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs) in the absence or presence of chemical growth factors. MyoD1 (A) and Myog (B), as skeletal muscle transcription factors, were mostly concentrated in the nuclei (arrows). (C) Myosin (fast skeletal). Undifferentiated stem cells were taken as a negative control, and the cell line L6 was considered as positive control (A-C, ×630).

    Article Snippet: Next, cells were blocked in 10% normal goat serum (Sigma-Aldrich) and incubated with mouse monoclonal anti-MyoD1 (1:100), mouse monoclonal anti-myogenin (1:100), or mouse monoclonal anti-myosin (fast skeletal, 1:100, Sigma-Aldrich) overnight at 4℃.

    Techniques: Expressing, Derivative Assay, Negative Control, Positive Control

    Stat3 promotes myogenic lineage progression in cultured satellite cells. ( a ) Left, representative images of satellite cells isolated by FACS, cultured in vitro and analyzed 5 d after isolation (green, pSTAT3; red, Myod1; blue, nuclei). Scale bar, 50 µm.

    Journal: Nature medicine

    Article Title: STAT3 signaling controls satellite cell expansion and skeletal muscle repair

    doi: 10.1038/nm.3656

    Figure Lengend Snippet: Stat3 promotes myogenic lineage progression in cultured satellite cells. ( a ) Left, representative images of satellite cells isolated by FACS, cultured in vitro and analyzed 5 d after isolation (green, pSTAT3; red, Myod1; blue, nuclei). Scale bar, 50 µm.

    Article Snippet: The antibodies used were as follows: rabbit anti–total Stat3 (79D7, Cell Signaling), rabbit anti-pStat3 (D3A7, Cell Signaling), rabbit anti-Gapdh (D16H11, Cell signaling) and mouse anti-Myod1 (cat#554130, BD Pharmingen).

    Techniques: Cell Culture, Isolation, FACS, In Vitro

    The effects of STAT3 manipulation are conserved in human myoblasts. ( a ) STAT3 and MYOD1 mRNA levels in human myoblasts infected with shSTAT3 lentivirus or shControl (shCtrl) at 96 h after infection ( n = 4). ( b ) Left, representative images of human myoblasts

    Journal: Nature medicine

    Article Title: STAT3 signaling controls satellite cell expansion and skeletal muscle repair

    doi: 10.1038/nm.3656

    Figure Lengend Snippet: The effects of STAT3 manipulation are conserved in human myoblasts. ( a ) STAT3 and MYOD1 mRNA levels in human myoblasts infected with shSTAT3 lentivirus or shControl (shCtrl) at 96 h after infection ( n = 4). ( b ) Left, representative images of human myoblasts

    Article Snippet: The antibodies used were as follows: rabbit anti–total Stat3 (79D7, Cell Signaling), rabbit anti-pStat3 (D3A7, Cell Signaling), rabbit anti-Gapdh (D16H11, Cell signaling) and mouse anti-Myod1 (cat#554130, BD Pharmingen).

    Techniques: Infection

    Expression of myogenic transcription factors in the delayed nerve repair model. The medial gastrocnemius muscles and the soleus muscles from ipsilateral operated sides were harvested and fast frozen in liquid nitrogen for subsequent qRT-PCR analysis of (A) MyoD and (B) myogenin after 0, 1, 3 and 6 month delayed repair with a donor nerve graft. Data are expressed relative to the immediate repairs. *P

    Journal: PLoS ONE

    Article Title: Investigation of the Expression of Myogenic Transcription Factors, microRNAs and Muscle-Specific E3 Ubiquitin Ligases in the Medial Gastrocnemius and Soleus Muscles following Peripheral Nerve Injury

    doi: 10.1371/journal.pone.0142699

    Figure Lengend Snippet: Expression of myogenic transcription factors in the delayed nerve repair model. The medial gastrocnemius muscles and the soleus muscles from ipsilateral operated sides were harvested and fast frozen in liquid nitrogen for subsequent qRT-PCR analysis of (A) MyoD and (B) myogenin after 0, 1, 3 and 6 month delayed repair with a donor nerve graft. Data are expressed relative to the immediate repairs. *P

    Article Snippet: 7μm thick sections were stained with rabbit anti-dystrophin antibody (GeneTex; 1:5000 dilution) and either mouse anti-MyoD antibody (BD Pharmingen; 1:200 dilution) or mouse anti-myogenin antibody (abcam; 1:200 dilution).

    Techniques: Expressing, Quantitative RT-PCR

    IL34 is required for myogenic lineage progression in cultured SCs. a Fresh SCs were seeded, cultured in growth medium, and then induced to differentiate for various amounts of time. The immunoblots presented here reveal the protein levels of IL34, p-STAT3, STAT3, and unrelated β-tubulin in cultured SCs at different time points after initiation of differentiation. b SCs were isolated from the hindlimbs of WT mice using FACS, cultured in growth medium for 3 days, and treated with shIL34 lentivirus or control shRNA (shScr) for 36 h. The positively infected SCs were then purified by FACS according to the intrinsic enhanced green fluorescent protein (EGFP) fluorescence expressed by the lentivirus. Purified infected SCs were cultured for further analysis. c Western blot analysis of the levels of IL34, p-STAT3, STAT3, and an unrelated protein (GAPDH) in WT cell cultures after shScr and shIL34 treatment. d Western blot analysis of the levels of MyHC, MyoD, Pax7, and an unrelated protein (GAPDH) in WT cell cultures after shScr and shIL34 treatment. e Representative merged photomicrographs of SCs stably infected with a lentivirus expressing shIL34 or shScr that were induced to differentiate for one day and stained for Pax7 and DAPI. Scale bar: 30 μm. f Quantification of the percentage of Pax7 + SCs after shScr and shIL34 infection. *** P

    Journal: Cell Death and Differentiation

    Article Title: Fate decision of satellite cell differentiation and self-renewal by miR-31-IL34 axis

    doi: 10.1038/s41418-019-0390-x

    Figure Lengend Snippet: IL34 is required for myogenic lineage progression in cultured SCs. a Fresh SCs were seeded, cultured in growth medium, and then induced to differentiate for various amounts of time. The immunoblots presented here reveal the protein levels of IL34, p-STAT3, STAT3, and unrelated β-tubulin in cultured SCs at different time points after initiation of differentiation. b SCs were isolated from the hindlimbs of WT mice using FACS, cultured in growth medium for 3 days, and treated with shIL34 lentivirus or control shRNA (shScr) for 36 h. The positively infected SCs were then purified by FACS according to the intrinsic enhanced green fluorescent protein (EGFP) fluorescence expressed by the lentivirus. Purified infected SCs were cultured for further analysis. c Western blot analysis of the levels of IL34, p-STAT3, STAT3, and an unrelated protein (GAPDH) in WT cell cultures after shScr and shIL34 treatment. d Western blot analysis of the levels of MyHC, MyoD, Pax7, and an unrelated protein (GAPDH) in WT cell cultures after shScr and shIL34 treatment. e Representative merged photomicrographs of SCs stably infected with a lentivirus expressing shIL34 or shScr that were induced to differentiate for one day and stained for Pax7 and DAPI. Scale bar: 30 μm. f Quantification of the percentage of Pax7 + SCs after shScr and shIL34 infection. *** P

    Article Snippet: The following primary antibodies were used: mouse anti-Pax7 (1:100, Developmental Studies Hybridoma Bank (DSHB), lowa City, IA), mouse anti-MyoD1 (1:100, DAKO), rabbit anti-MyoD (1:100, Santa Cruz, sc-760), mouse anti-MyoD (1:100, Santa Cruz, sc-32758), mouse anti-embryonic Myosin Heavy Chain BF-45/F1.652 (1:40, DSHB, lowa City, IA), rabbit anti-myogenin (1:200, Santa Cruz, sc-576), rabbit anti-laminin (1:1000, Sigma-Aldrich, L9393), and rabbit anti-Ki67 (1:500, Invitrogen).

    Techniques: Cell Culture, Western Blot, Isolation, Mouse Assay, FACS, shRNA, Infection, Purification, Fluorescence, Stable Transfection, Expressing, Staining

    Inactivation of miR-31 promotes the myogenic lineage progression of SCs. a WT and miR-31-KO SCs were cultured in growth medium for 4 days and then switched to differentiation medium and cultured for 1 day. The cells were then fixed and labeled with anti-MyoD and anti-Ki67 antibodies. DAPI was used to identify nuclei. Representative individual and overlaid images of WT and miR-31-KO cultures after labeling with MyoD, Ki67, and DAPI. Scale bars: 30 μm. b Percentage of MyoD + Ki67 − cells (normalized to MyoD + cells) in WT and miR-31-KO cell cultures. ** P

    Journal: Cell Death and Differentiation

    Article Title: Fate decision of satellite cell differentiation and self-renewal by miR-31-IL34 axis

    doi: 10.1038/s41418-019-0390-x

    Figure Lengend Snippet: Inactivation of miR-31 promotes the myogenic lineage progression of SCs. a WT and miR-31-KO SCs were cultured in growth medium for 4 days and then switched to differentiation medium and cultured for 1 day. The cells were then fixed and labeled with anti-MyoD and anti-Ki67 antibodies. DAPI was used to identify nuclei. Representative individual and overlaid images of WT and miR-31-KO cultures after labeling with MyoD, Ki67, and DAPI. Scale bars: 30 μm. b Percentage of MyoD + Ki67 − cells (normalized to MyoD + cells) in WT and miR-31-KO cell cultures. ** P

    Article Snippet: The following primary antibodies were used: mouse anti-Pax7 (1:100, Developmental Studies Hybridoma Bank (DSHB), lowa City, IA), mouse anti-MyoD1 (1:100, DAKO), rabbit anti-MyoD (1:100, Santa Cruz, sc-760), mouse anti-MyoD (1:100, Santa Cruz, sc-32758), mouse anti-embryonic Myosin Heavy Chain BF-45/F1.652 (1:40, DSHB, lowa City, IA), rabbit anti-myogenin (1:200, Santa Cruz, sc-576), rabbit anti-laminin (1:1000, Sigma-Aldrich, L9393), and rabbit anti-Ki67 (1:500, Invitrogen).

    Techniques: Cell Culture, Labeling

    Successful myotube differentiation of 3-deazaneplanocin A hydrochloride (DZNep)-treated MYOD1 -urine-derived cells (UDCs) derived from healthy individuals. ( A ) Representative images of immunocytochemistry for MyHC (upper) and DYSTROPHIN (lower) in MYOD1 -UDCs from healthy children and adults 14 days after differentiation. HI: healthy individual. #1: 8-year-old male; #2: 13-year-old male; #3: 33-year-old male; #4: 38-year-old male. Scale bar: 100 μm. ( B ) Comparison of fusion index with and without DZNep treatment. The fusion index was calculated as a percentage of nuclei inside the MyHC positive myotubes using randomly selected three pictures from four healthy individuals respectively. The Mann-Whitney test was used for comparisons; ***P

    Journal: Scientific Reports

    Article Title: Modelling Duchenne muscular dystrophy in MYOD1-converted urine-derived cells treated with 3-deazaneplanocin A hydrochloride

    doi: 10.1038/s41598-019-40421-z

    Figure Lengend Snippet: Successful myotube differentiation of 3-deazaneplanocin A hydrochloride (DZNep)-treated MYOD1 -urine-derived cells (UDCs) derived from healthy individuals. ( A ) Representative images of immunocytochemistry for MyHC (upper) and DYSTROPHIN (lower) in MYOD1 -UDCs from healthy children and adults 14 days after differentiation. HI: healthy individual. #1: 8-year-old male; #2: 13-year-old male; #3: 33-year-old male; #4: 38-year-old male. Scale bar: 100 μm. ( B ) Comparison of fusion index with and without DZNep treatment. The fusion index was calculated as a percentage of nuclei inside the MyHC positive myotubes using randomly selected three pictures from four healthy individuals respectively. The Mann-Whitney test was used for comparisons; ***P

    Article Snippet: The following primary antibodies were used: mouse anti-MYOD1 (1:200, Santa Cruz; sc-32758), mouse anti-myogenin (1:200, Santa Cruz; sc-12732), mouse anti-dystrophin (1:30, Leica, NCL-DYS1), mouse anti-myosin heavy chain (1:50, R & D; MAB4470), mouse anti-α-Actinin (1:1000, Sigma; A7811), mouse anti-desmin (1:800, Sigma; D1033), and mouse anti-β-dystroglycan (1:100, Leica, NCL-b-DG).

    Techniques: Derivative Assay, Immunocytochemistry, MANN-WHITNEY

    Successful evaluation of exon-skipping in urine-derived cells (UDCs) from patients with exon 45–54 deletions. RT-PCR analysis of DYSTROPHIN after phosphorodiamidate morpholino oligomer (PMO) treatment in ( A ) 3-deazaneplanocin A hydrochloride (DZNep)-treated MYOD1 -UDCs and ( B ) MYOD1 -converted fibroblasts ( MYOD1 - Fibs) derived from a Duchenne muscular dystrophy (DMD) patient with an exon 45–54 deletion. DZNep-treated MYOD1 -UDCs and MYOD1 - Fibs were also treated with control antisense at 5 μM as controls. The upper bands are unskipped products (Δ45–54) that remain out of the reading frame. The lower bands are exon 44-skipped products (Δ44–54) that restore the open reading frame. Skipping efficiency was calculated as (exon 44-skipped transcript molarity)/(native + exon 44-skipped transcript molarity (marked with arrows)) ×100% using MultiNA. One-way ANOVA followed by Bonferroni’s post hoc test was used to compare the skipping efficiencies; n = 3, for each group; ****P

    Journal: Scientific Reports

    Article Title: Modelling Duchenne muscular dystrophy in MYOD1-converted urine-derived cells treated with 3-deazaneplanocin A hydrochloride

    doi: 10.1038/s41598-019-40421-z

    Figure Lengend Snippet: Successful evaluation of exon-skipping in urine-derived cells (UDCs) from patients with exon 45–54 deletions. RT-PCR analysis of DYSTROPHIN after phosphorodiamidate morpholino oligomer (PMO) treatment in ( A ) 3-deazaneplanocin A hydrochloride (DZNep)-treated MYOD1 -UDCs and ( B ) MYOD1 -converted fibroblasts ( MYOD1 - Fibs) derived from a Duchenne muscular dystrophy (DMD) patient with an exon 45–54 deletion. DZNep-treated MYOD1 -UDCs and MYOD1 - Fibs were also treated with control antisense at 5 μM as controls. The upper bands are unskipped products (Δ45–54) that remain out of the reading frame. The lower bands are exon 44-skipped products (Δ44–54) that restore the open reading frame. Skipping efficiency was calculated as (exon 44-skipped transcript molarity)/(native + exon 44-skipped transcript molarity (marked with arrows)) ×100% using MultiNA. One-way ANOVA followed by Bonferroni’s post hoc test was used to compare the skipping efficiencies; n = 3, for each group; ****P

    Article Snippet: The following primary antibodies were used: mouse anti-MYOD1 (1:200, Santa Cruz; sc-32758), mouse anti-myogenin (1:200, Santa Cruz; sc-12732), mouse anti-dystrophin (1:30, Leica, NCL-DYS1), mouse anti-myosin heavy chain (1:50, R & D; MAB4470), mouse anti-α-Actinin (1:1000, Sigma; A7811), mouse anti-desmin (1:800, Sigma; D1033), and mouse anti-β-dystroglycan (1:100, Leica, NCL-b-DG).

    Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction

    Successful evaluation of exon-skipping using urine-derived cells (UDCs) from patients with various exon deletions in a hotspot region. ( A ) RT-PCR analysis of DYSTROPHIN after phosphorodiamidate morpholino oligomer (PMO) treatment in 3-deazaneplanocin A hydrochloride (DZNep)-treated MYOD1 -UDCs derived from Duchenne muscular dystrophy (DMD) patients with exon 50, 51, and 54 deletions are shown. Exon 50, 51, or 54 deletions were restored in-frame by skipping exons 51, 50, or 55, respectively. Exon-skipping efficiency was calculated as (exon-skipped transcript molarity)/(native + intermediate + exon-skipped transcript molarity (marked with arrows)) × 100% using MultiNA. One-way ANOVA followed by Bonferroni’s post hoc test was used to compare the skipping efficiency between patient-derived DZNep-treated MYOD1 -UDCs; n = 3, each. *P

    Journal: Scientific Reports

    Article Title: Modelling Duchenne muscular dystrophy in MYOD1-converted urine-derived cells treated with 3-deazaneplanocin A hydrochloride

    doi: 10.1038/s41598-019-40421-z

    Figure Lengend Snippet: Successful evaluation of exon-skipping using urine-derived cells (UDCs) from patients with various exon deletions in a hotspot region. ( A ) RT-PCR analysis of DYSTROPHIN after phosphorodiamidate morpholino oligomer (PMO) treatment in 3-deazaneplanocin A hydrochloride (DZNep)-treated MYOD1 -UDCs derived from Duchenne muscular dystrophy (DMD) patients with exon 50, 51, and 54 deletions are shown. Exon 50, 51, or 54 deletions were restored in-frame by skipping exons 51, 50, or 55, respectively. Exon-skipping efficiency was calculated as (exon-skipped transcript molarity)/(native + intermediate + exon-skipped transcript molarity (marked with arrows)) × 100% using MultiNA. One-way ANOVA followed by Bonferroni’s post hoc test was used to compare the skipping efficiency between patient-derived DZNep-treated MYOD1 -UDCs; n = 3, each. *P

    Article Snippet: The following primary antibodies were used: mouse anti-MYOD1 (1:200, Santa Cruz; sc-32758), mouse anti-myogenin (1:200, Santa Cruz; sc-12732), mouse anti-dystrophin (1:30, Leica, NCL-DYS1), mouse anti-myosin heavy chain (1:50, R & D; MAB4470), mouse anti-α-Actinin (1:1000, Sigma; A7811), mouse anti-desmin (1:800, Sigma; D1033), and mouse anti-β-dystroglycan (1:100, Leica, NCL-b-DG).

    Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction

    MYOD1 and 3-deazaneplanocin A hydrochloride promote the direct-reprogramming of urine-derived cells into myotubes. ( A ) Schema of the retroviral vector with the MYOD1 and puromycin resistance genes. The TRE3GS promoter is activated in the presence of doxycycline. ( B ) Schematic diagram of the transduction of the viral vector. ( C ) Results of drug screening using a chemical library (Sigma; S990043-EPI1). Representative data are shown. The area of myosin heavy chain (MyHC)-positivity was determined by fluorescence microscopy at 14 th day after differentiation. Urine-derived cells (UDCs) were pre-treated with various chemical compounds for initial 3 days after differentiation (final concentrations = 0.1, 1, and 10 μM). The Kruskal-Wallis test followed by a Dunn’s post hoc test was used for statistical analysis; *P

    Journal: Scientific Reports

    Article Title: Modelling Duchenne muscular dystrophy in MYOD1-converted urine-derived cells treated with 3-deazaneplanocin A hydrochloride

    doi: 10.1038/s41598-019-40421-z

    Figure Lengend Snippet: MYOD1 and 3-deazaneplanocin A hydrochloride promote the direct-reprogramming of urine-derived cells into myotubes. ( A ) Schema of the retroviral vector with the MYOD1 and puromycin resistance genes. The TRE3GS promoter is activated in the presence of doxycycline. ( B ) Schematic diagram of the transduction of the viral vector. ( C ) Results of drug screening using a chemical library (Sigma; S990043-EPI1). Representative data are shown. The area of myosin heavy chain (MyHC)-positivity was determined by fluorescence microscopy at 14 th day after differentiation. Urine-derived cells (UDCs) were pre-treated with various chemical compounds for initial 3 days after differentiation (final concentrations = 0.1, 1, and 10 μM). The Kruskal-Wallis test followed by a Dunn’s post hoc test was used for statistical analysis; *P

    Article Snippet: The following primary antibodies were used: mouse anti-MYOD1 (1:200, Santa Cruz; sc-32758), mouse anti-myogenin (1:200, Santa Cruz; sc-12732), mouse anti-dystrophin (1:30, Leica, NCL-DYS1), mouse anti-myosin heavy chain (1:50, R & D; MAB4470), mouse anti-α-Actinin (1:1000, Sigma; A7811), mouse anti-desmin (1:800, Sigma; D1033), and mouse anti-β-dystroglycan (1:100, Leica, NCL-b-DG).

    Techniques: Derivative Assay, Plasmid Preparation, Transduction, Fluorescence, Microscopy

    Marker expression in SC clones cultured under normoxia and hypoxia. Immunofluorescence analyses of LPC and HPC cultured for 5 days at 20% and 2% O 2 revealed differences in the expression of MyoD, Myf5 (only at 2% O 2 ) and CD34. Phase-contrast and corresponding immunostaining examples for the specific marker (in red) merged with DAPI (left, scale bar = 100 µm, insets with higher magnification). Graphs indicate the percentage of marker-positive cells per clone (right, mean±SEM, * p

    Journal: PLoS ONE

    Article Title: Hypoxia Increases Mouse Satellite Cell Clone Proliferation Maintaining both In Vitro and In Vivo Heterogeneity and Myogenic Potential

    doi: 10.1371/journal.pone.0049860

    Figure Lengend Snippet: Marker expression in SC clones cultured under normoxia and hypoxia. Immunofluorescence analyses of LPC and HPC cultured for 5 days at 20% and 2% O 2 revealed differences in the expression of MyoD, Myf5 (only at 2% O 2 ) and CD34. Phase-contrast and corresponding immunostaining examples for the specific marker (in red) merged with DAPI (left, scale bar = 100 µm, insets with higher magnification). Graphs indicate the percentage of marker-positive cells per clone (right, mean±SEM, * p

    Article Snippet: 1∶20), rabbit anti-mouse Myf5 (Santa Cruz Biotechnology, dilution 1∶20), rabbit anti-mouse MyoD (Santa Cruz, dil.

    Techniques: Marker, Expressing, Clone Assay, Cell Culture, Immunofluorescence, Immunostaining

    Paracrine WISP1 secretion from young FAPs stimulates the myogenic commitment of aged MuSCs (A) Experimental overview of the in-vivo transplantation of FAPs in WISP1−/− recipient mice. (B and C) Quantification and representative images of Pax7+/MyoD+ MuSCs by immunofluorescence in muscle cross-sections of WISP1−/− mice at 4dpi, after intra-muscular injection of vehicle or FAPs freshly isolated from young, aged or WISP1−/− mice at 1 dpi. n≥3 mice per condition. (D) Experimental overview of the in-vivo transplantation of FAPs in aged WT recipient mice. (E) Quantification of the number of Pax7+/MyoD+ MuSCs by immunofluorescence in muscle cross-sections of aged WT mice at 4dpi, after intra-muscular injection of vehicle or FAPs freshly isolated from young, aged or WISP1−/− mice at 1 dpi. n≥4 mice per condition. (B and E) Data are represented as means ± S.E.M. p-values are *

    Journal: Cell stem cell

    Article Title: Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors

    doi: 10.1016/j.stem.2018.12.014

    Figure Lengend Snippet: Paracrine WISP1 secretion from young FAPs stimulates the myogenic commitment of aged MuSCs (A) Experimental overview of the in-vivo transplantation of FAPs in WISP1−/− recipient mice. (B and C) Quantification and representative images of Pax7+/MyoD+ MuSCs by immunofluorescence in muscle cross-sections of WISP1−/− mice at 4dpi, after intra-muscular injection of vehicle or FAPs freshly isolated from young, aged or WISP1−/− mice at 1 dpi. n≥3 mice per condition. (D) Experimental overview of the in-vivo transplantation of FAPs in aged WT recipient mice. (E) Quantification of the number of Pax7+/MyoD+ MuSCs by immunofluorescence in muscle cross-sections of aged WT mice at 4dpi, after intra-muscular injection of vehicle or FAPs freshly isolated from young, aged or WISP1−/− mice at 1 dpi. n≥4 mice per condition. (B and E) Data are represented as means ± S.E.M. p-values are *

    Article Snippet: Mouse anti-mouse Pax7 (DHSB, purified), rabbit-anti mouse MyoD antibody (Santa-Cruz #sc-304 or Abcam # ab198251), rabbit-anti mouse Ki67 (Abcam #ab15580) and chicken anti-human laminin (Life Span Bioscience #LC-C96142-100) antibodies were used at 2.5 μg/ml, 1/100, 1/100 and 1/200 in blocking solution, respectively.

    Techniques: In Vivo, Transplantation Assay, Mouse Assay, Immunofluorescence, Injection, Isolation

    Myogenic marker expression of isolated SC/MPC populations. Flow cytometric analysis of immunofluorescence-stained SC/MPC from SM and LD muscle cultured for 4 days ( a ) and 8 days ( b ) in growth medium. The cells were immunostained for Pax7, MyoD1, MyoG, and Desmin. Percentages of positive cells in each sample are presented as Box-Whisker plots with the median and the maximum 1.5 of the interquartile range (Q1-Q3). Outliers are included in the figure as circles but were excluded from statistical analysis. All populations expressed the selected myogenic markers, whereas a higher proportion of cells were positive for MyoG and Desmin, which are markers for terminally committed myogenic cells. Statistical analysis was performed by ANOVA (with the Holm-Sidak method as a pairwise multiple comparison procedure) or the Kruskal-Wallis One-Way ANOVA on Ranks (with Dunn’s method as a pairwise multiple comparison procedure), *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, n = 4–9 animals

    Journal: BMC Cell Biology

    Article Title: Separation of functionally divergent muscle precursor cell populations from porcine juvenile muscles by discontinuous Percoll density gradient centrifugation

    doi: 10.1186/s12860-018-0156-1

    Figure Lengend Snippet: Myogenic marker expression of isolated SC/MPC populations. Flow cytometric analysis of immunofluorescence-stained SC/MPC from SM and LD muscle cultured for 4 days ( a ) and 8 days ( b ) in growth medium. The cells were immunostained for Pax7, MyoD1, MyoG, and Desmin. Percentages of positive cells in each sample are presented as Box-Whisker plots with the median and the maximum 1.5 of the interquartile range (Q1-Q3). Outliers are included in the figure as circles but were excluded from statistical analysis. All populations expressed the selected myogenic markers, whereas a higher proportion of cells were positive for MyoG and Desmin, which are markers for terminally committed myogenic cells. Statistical analysis was performed by ANOVA (with the Holm-Sidak method as a pairwise multiple comparison procedure) or the Kruskal-Wallis One-Way ANOVA on Ranks (with Dunn’s method as a pairwise multiple comparison procedure), *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, n = 4–9 animals

    Article Snippet: Following fixation, cells were permeabilized with 0.1% (Desmin, MyoG) or 0.5% (Pax7, MyoD1) Triton X-100 in PBS, blocked with normal rabbit serum, and incubated overnight with the respective primary antibody: mouse anti-Desmin (clone D-33, DAKO, 1:80), mouse anti-Pax7 (Developmental Studies Hydridoma Bank, 1:50), mouse anti-MyoG (clone F5D, abcam, 1:50), or mouse anti-MyoD1 (clone 5.2F, abcam, 1:200).

    Techniques: Marker, Expressing, Isolation, Flow Cytometry, Immunofluorescence, Staining, Cell Culture, Whisker Assay

    Schematic of C2C12 Differentiation (A) In the C2C12 cell culture system, upon removal of serum, proliferating myoblasts can de-differentiate into quiescent reserve cells. Reserve cells are capable of differentiating into myoblasts with the addition of serum. Myoblasts are Pax7 + /MyoD + ; whereas, reserve cells are Pax7 + /MyoD − . Cells are guided to the reserve cell fate by expression and activation of the Notch receptor, which gives elevated Hes and Hey gene expression and maintains the self-renewing state. (B) Upon serum removal, C2C12 cells also differentiate into myocytes, which are Pax7/MyoD + . Myocytes lose their proliferative capabilities, express myogenin (MyoG), followed by myosin heavy chain (MHC), and ultimately fuse to form syncytial myotubes.

    Journal: Toxicology

    Article Title: Methylmercury exposure causes a persistent inhibition of myogenin expression and C2C12 myoblast differentiation

    doi: 10.1016/j.tox.2017.11.002

    Figure Lengend Snippet: Schematic of C2C12 Differentiation (A) In the C2C12 cell culture system, upon removal of serum, proliferating myoblasts can de-differentiate into quiescent reserve cells. Reserve cells are capable of differentiating into myoblasts with the addition of serum. Myoblasts are Pax7 + /MyoD + ; whereas, reserve cells are Pax7 + /MyoD − . Cells are guided to the reserve cell fate by expression and activation of the Notch receptor, which gives elevated Hes and Hey gene expression and maintains the self-renewing state. (B) Upon serum removal, C2C12 cells also differentiate into myocytes, which are Pax7/MyoD + . Myocytes lose their proliferative capabilities, express myogenin (MyoG), followed by myosin heavy chain (MHC), and ultimately fuse to form syncytial myotubes.

    Article Snippet: Primary antibodies used were: mouse monoclonal anti-MyoG (Developmental Studies Hybridoma Bank (DSHB), F5D) diluted 1/50, mouse monoclonal anti-MHC (DSHB, MF20), diluted 1/50, rabbit polyclonal anti-Pax7 (Abcam, ab187339) diluted 1/100, mouse monoclonal anti-MyoD (BD Biosciences, 5.8A) diluted 1/100, and rat monoclonal anti-Ki67 (Biolegend, 16A8) diluted 1/200.

    Techniques: Cell Culture, Expressing, Activation Assay

    Effect of MeHg on Expression of Myogenic Differentiation Genes within the First 24-hours of MeHg Exposure Gene transcript levels, with and without 2.5μM MeHg of (A) Pax7 , (B) MyoD , and (C) MyoG were analyzed by RT-qPCR at the indicated time points. Each data point is normalized to the undifferentiated cell control (time 0). (n=3; two-way ANOVA with Sidak’s test for multiple comparisons, *p≤0.05, **p≤0.01, ****p

    Journal: Toxicology

    Article Title: Methylmercury exposure causes a persistent inhibition of myogenin expression and C2C12 myoblast differentiation

    doi: 10.1016/j.tox.2017.11.002

    Figure Lengend Snippet: Effect of MeHg on Expression of Myogenic Differentiation Genes within the First 24-hours of MeHg Exposure Gene transcript levels, with and without 2.5μM MeHg of (A) Pax7 , (B) MyoD , and (C) MyoG were analyzed by RT-qPCR at the indicated time points. Each data point is normalized to the undifferentiated cell control (time 0). (n=3; two-way ANOVA with Sidak’s test for multiple comparisons, *p≤0.05, **p≤0.01, ****p

    Article Snippet: Primary antibodies used were: mouse monoclonal anti-MyoG (Developmental Studies Hybridoma Bank (DSHB), F5D) diluted 1/50, mouse monoclonal anti-MHC (DSHB, MF20), diluted 1/50, rabbit polyclonal anti-Pax7 (Abcam, ab187339) diluted 1/100, mouse monoclonal anti-MyoD (BD Biosciences, 5.8A) diluted 1/100, and rat monoclonal anti-Ki67 (Biolegend, 16A8) diluted 1/200.

    Techniques: Expressing, Quantitative RT-PCR

    Myoblast Differentiation with 24-hour MeHg Exposure and Recovery Immunostaining of C2C12 cells with MyoG antibody (green) after a 24-hour exposure with (A) 0μM MeHg and (B) 2.5μM MeHg followed by 3-day recovery. Immunostaining of C2C12 cells with Pax7 (green) and MyoD (red) after a 24-hour exposure with (C) 0μM MeHg or (D) 2.5μM MeHg followed by 3-day recovery. (E) Quantification of the number of nuclei that expressed MyoG, expressed as a percentage of 0μM control, across 5 doses of MeHg (n=3, one-way ANOVA with Tukey’s test for multiple comparisons, compared to 0μM exposure, **p≤0.01, *** p

    Journal: Toxicology

    Article Title: Methylmercury exposure causes a persistent inhibition of myogenin expression and C2C12 myoblast differentiation

    doi: 10.1016/j.tox.2017.11.002

    Figure Lengend Snippet: Myoblast Differentiation with 24-hour MeHg Exposure and Recovery Immunostaining of C2C12 cells with MyoG antibody (green) after a 24-hour exposure with (A) 0μM MeHg and (B) 2.5μM MeHg followed by 3-day recovery. Immunostaining of C2C12 cells with Pax7 (green) and MyoD (red) after a 24-hour exposure with (C) 0μM MeHg or (D) 2.5μM MeHg followed by 3-day recovery. (E) Quantification of the number of nuclei that expressed MyoG, expressed as a percentage of 0μM control, across 5 doses of MeHg (n=3, one-way ANOVA with Tukey’s test for multiple comparisons, compared to 0μM exposure, **p≤0.01, *** p

    Article Snippet: Primary antibodies used were: mouse monoclonal anti-MyoG (Developmental Studies Hybridoma Bank (DSHB), F5D) diluted 1/50, mouse monoclonal anti-MHC (DSHB, MF20), diluted 1/50, rabbit polyclonal anti-Pax7 (Abcam, ab187339) diluted 1/100, mouse monoclonal anti-MyoD (BD Biosciences, 5.8A) diluted 1/100, and rat monoclonal anti-Ki67 (Biolegend, 16A8) diluted 1/200.

    Techniques: Immunostaining

    Analysis of gelatinase activity in satellite cells of trained Sol muscle (5 days of running). Confocal maximum projection images demonstrating the absence of gelatinolytic activity ( a , d , green ) in M-cadherin-immunoreactive ( b , d , red , arrowheads ) quiescent satellite cell ( asterisk ) lying adjacent to a myonucleus containing strong gelatinase activity; nuclei are counterstained with TOPRO dye ( c , d , blue ). Scale bar 20 μm. A group of cells having strong gelatinase activity ( e , g , green ) and the immunoreactivity of the satellite cell marker PAX7 ( f , g , red ), lying within the space formerly occupied by the necrotic fiber; the expression of the specific marker, and the positioning of the cells away from the sarcolemma identifies them as activated satellite cells or myoblasts. An arrow points to the quiescent PAX7-positive satellite cell adjacent to the muscle fiber; this cell does not contain the gelatinase activity. The images are confocal maximum projections. Scale bar 10 μm. A group of strongly gelatinase-postive cells ( h , green ) immunoreactive for the specific marker of activated satellite cell/myoblasts MyoD ( i , blue ), expressing MMP-9-IR ( j , red ). Scale bar 25 μm. Confocal maximum projection images demonstrating the absence of MMP-2-IR ( m , n , red ) in gelatinase-positive ( k , n , green ), MyoD-immunoreactive ( l , n , blue ) activated satellite cells/myoblasts. Scale bar 25 μm. o – r Confocal maximum projection images demonstrating that gelatinase-positive ( o , r , green ), PAX7-immunoreactive ( p , r , blue ) are distinct from CD45-immunoreactive macrophage-like cells. Scale bar 25 μm

    Journal: Histochemistry and Cell Biology

    Article Title: Fine-structural distribution of MMP-2 and MMP-9 activities in the rat skeletal muscle upon training: a study by high-resolution in situ zymography

    doi: 10.1007/s00418-012-0940-5

    Figure Lengend Snippet: Analysis of gelatinase activity in satellite cells of trained Sol muscle (5 days of running). Confocal maximum projection images demonstrating the absence of gelatinolytic activity ( a , d , green ) in M-cadherin-immunoreactive ( b , d , red , arrowheads ) quiescent satellite cell ( asterisk ) lying adjacent to a myonucleus containing strong gelatinase activity; nuclei are counterstained with TOPRO dye ( c , d , blue ). Scale bar 20 μm. A group of cells having strong gelatinase activity ( e , g , green ) and the immunoreactivity of the satellite cell marker PAX7 ( f , g , red ), lying within the space formerly occupied by the necrotic fiber; the expression of the specific marker, and the positioning of the cells away from the sarcolemma identifies them as activated satellite cells or myoblasts. An arrow points to the quiescent PAX7-positive satellite cell adjacent to the muscle fiber; this cell does not contain the gelatinase activity. The images are confocal maximum projections. Scale bar 10 μm. A group of strongly gelatinase-postive cells ( h , green ) immunoreactive for the specific marker of activated satellite cell/myoblasts MyoD ( i , blue ), expressing MMP-9-IR ( j , red ). Scale bar 25 μm. Confocal maximum projection images demonstrating the absence of MMP-2-IR ( m , n , red ) in gelatinase-positive ( k , n , green ), MyoD-immunoreactive ( l , n , blue ) activated satellite cells/myoblasts. Scale bar 25 μm. o – r Confocal maximum projection images demonstrating that gelatinase-positive ( o , r , green ), PAX7-immunoreactive ( p , r , blue ) are distinct from CD45-immunoreactive macrophage-like cells. Scale bar 25 μm

    Article Snippet: To visualize selected epitopes, specific antibodies were used: polyclonal rabbit anti-MMP-9 (Torrey Pines Biolabs), diluted 1:100, polyclonal rabbit anti-MMP-2, 1:200 (Santa Cruz), monoclonal mouse anti-Pax-7, 1:500 (Developmental Studies Hybridoma Bank, University of Iowa), monoclonal mouse M-Cadherin, 1:100 (BD Biosciences), monoclonal mouse anti-MyoD, 1:100 (BD Biosciences), monoclonal mouse anti-CD45 biotinolated, 1:100 (Abcam), monoclonal mouse anti-desmin, 1:25 (Sigma), monoclonal mouse anti-β-Dystroglican, 1:10 (Novocastra), polyclonal rabbit anti-RNA PolII (phospho S2) (1:100, Abcam).

    Techniques: Activity Assay, Marker, Expressing

    Myogenic progression in S1PR3 -null satellite cells is relatively normal. EDL myofibres and their associated satellite cells were isolated from either age-matched wild-type ( S1PR3 +/+ ) or S1PR3- null ( S1PR3 −/− ) mice and cultured in proliferation medium for 72 h. (A)–(C) Samples were taken at 24 h intervals, fixed and co-immunostained for Pax7, MyoD and Myogenin, which showed a general increase in each population when S1PR3 was absent, consistent with enhanced proliferation. (D) When expressed as a ratio though, the proportions of cells expressing Pax7, MyoD or myogenin were similar between wildtype and S1PR3 -null mice. (E) and (F) Expanded satellite cell-derived myoblasts were also plated at high confluency and then cultured in differentiation medium for 48 h before immunostaining for Myosin Heavy Chain (MyHC) and counterstaining with DAPI. Counting the number of nuclei within MyHC+ve myotubes ( > 2 nuclei) to determine the fusion index revealed that more satellite cells from S1PR3 -null mice had differentiated and fused than from control mice ((E), quantified (F)). Values are expressed as mean±SEM from multiple samples from at least three mice ( n =3/4) where an asterisk denotes significantly different ( p

    Journal: Developmental Biology

    Article Title: Sphingosine-1-phosphate receptor 3 influences cell cycle progression in muscle satellite cells

    doi: 10.1016/j.ydbio.2013.07.006

    Figure Lengend Snippet: Myogenic progression in S1PR3 -null satellite cells is relatively normal. EDL myofibres and their associated satellite cells were isolated from either age-matched wild-type ( S1PR3 +/+ ) or S1PR3- null ( S1PR3 −/− ) mice and cultured in proliferation medium for 72 h. (A)–(C) Samples were taken at 24 h intervals, fixed and co-immunostained for Pax7, MyoD and Myogenin, which showed a general increase in each population when S1PR3 was absent, consistent with enhanced proliferation. (D) When expressed as a ratio though, the proportions of cells expressing Pax7, MyoD or myogenin were similar between wildtype and S1PR3 -null mice. (E) and (F) Expanded satellite cell-derived myoblasts were also plated at high confluency and then cultured in differentiation medium for 48 h before immunostaining for Myosin Heavy Chain (MyHC) and counterstaining with DAPI. Counting the number of nuclei within MyHC+ve myotubes ( > 2 nuclei) to determine the fusion index revealed that more satellite cells from S1PR3 -null mice had differentiated and fused than from control mice ((E), quantified (F)). Values are expressed as mean±SEM from multiple samples from at least three mice ( n =3/4) where an asterisk denotes significantly different ( p

    Article Snippet: Immunocytochemistry Primary antibodies used were: mouse monoclonal anti-Pax7 and anti-myogenin clone F5D (Developmental Studies Hybridoma Bank, Iowa, USA); mouse monoclonal anti-MyoD clone 5.8A (DakoCytomation); rabbit polyclonal anti-MyoD (Santa Cruz Biotechnology); polyclonal rabbit anti-GFP (Invitrogen); rat monoclonal anti-Ki67 (Dako Ltd., Kyoto, Japan).

    Techniques: Isolation, Mouse Assay, Cell Culture, Expressing, Derivative Assay, Immunostaining

    Pax3/Pax7 transcriptional activity is required for myogenin expression. To test the effects of perturbing Pax3 and Pax7 function on myogenicity, cells were infected with retroviral constructs encoding Pax3, Pax7, Pax3DN or Pax7DN, together with empty vector serving as control. Cells underwent further culture and were then co-immunostained for either eGFP (green) and MyoD (red), or eGFP (green) and myogenin (red). C2C12 cells infected with control RV (a), Pax3 RV (b), Pax7 RV (c), Pax3DN RV (d) and Pax7DN RV (e) and co-immunostained for eGFP and myogenin 120 h after infection. Constitutive Pax3 or Pax7 expression resulted in more infected cells (eGFP+ve) with MyoD, while constitutive Pax3DN or Pax7DN caused less eGFP+ve cells to co-express MyoD and myogenin (quantified in p after 72 h). Satellite cells were infected with control RV (f), Pax3 RV (g), Pax7 RV (h), Pax3DN RV (i) and Pax7DN RV (j) while retained in their niche on the myofibre and cultured before co-immunostaining for eGFP (green) and myogenin (red). The presence of constitutively expressed Pax3 (g) or Pax7 (h) was compatible with myogenin expression (arrows), but there were reduced numbers of infected eGFP+ve satellite cell progeny containing myogenin (quantified in q). Many infected GFP+ve cells expressing Pax3DN (i - green - arrow) and Pax7DN (j - green - arrow) did not contain myogenin (i and j - red) (quantified in q). Similar results were obtained when plated satellite cell-derived myoblasts were infected with control RV (k), Pax3 RV (l), Pax7 RV (m), Pax3DN RV (n) and Pax7DN RV (o) and co-stained for eGFP (green) and myogenin (red), 72 h post infection (quantified in r). Counterstaining with DAPI was used to identify all nuclei present. Scale bar for each row equals 40 µm, except for k–o where it represents 100 µm. Values are population means±SEM of at least 3 independent experiments, where an asterisk denotes significant difference at p

    Journal: PLoS ONE

    Article Title: Integrated Functions of Pax3 and Pax7 in the Regulation of Proliferation, Cell Size and Myogenic Differentiation

    doi: 10.1371/journal.pone.0004475

    Figure Lengend Snippet: Pax3/Pax7 transcriptional activity is required for myogenin expression. To test the effects of perturbing Pax3 and Pax7 function on myogenicity, cells were infected with retroviral constructs encoding Pax3, Pax7, Pax3DN or Pax7DN, together with empty vector serving as control. Cells underwent further culture and were then co-immunostained for either eGFP (green) and MyoD (red), or eGFP (green) and myogenin (red). C2C12 cells infected with control RV (a), Pax3 RV (b), Pax7 RV (c), Pax3DN RV (d) and Pax7DN RV (e) and co-immunostained for eGFP and myogenin 120 h after infection. Constitutive Pax3 or Pax7 expression resulted in more infected cells (eGFP+ve) with MyoD, while constitutive Pax3DN or Pax7DN caused less eGFP+ve cells to co-express MyoD and myogenin (quantified in p after 72 h). Satellite cells were infected with control RV (f), Pax3 RV (g), Pax7 RV (h), Pax3DN RV (i) and Pax7DN RV (j) while retained in their niche on the myofibre and cultured before co-immunostaining for eGFP (green) and myogenin (red). The presence of constitutively expressed Pax3 (g) or Pax7 (h) was compatible with myogenin expression (arrows), but there were reduced numbers of infected eGFP+ve satellite cell progeny containing myogenin (quantified in q). Many infected GFP+ve cells expressing Pax3DN (i - green - arrow) and Pax7DN (j - green - arrow) did not contain myogenin (i and j - red) (quantified in q). Similar results were obtained when plated satellite cell-derived myoblasts were infected with control RV (k), Pax3 RV (l), Pax7 RV (m), Pax3DN RV (n) and Pax7DN RV (o) and co-stained for eGFP (green) and myogenin (red), 72 h post infection (quantified in r). Counterstaining with DAPI was used to identify all nuclei present. Scale bar for each row equals 40 µm, except for k–o where it represents 100 µm. Values are population means±SEM of at least 3 independent experiments, where an asterisk denotes significant difference at p

    Article Snippet: Primary antibodies used were monoclonal rat anti-BrdU clone BU1/75 (Abcam), monoclonal mouse anti-myogenin clone F5D (Developmental Studies Hybridoma Bank), polyclonal rabbit anti-myogenin (Santa Cruz), monoclonal mouse anti-MyoD clone 5.8A (DakoCytomation), polyclonal rabbit anti-MyoD (Santa Cruz), monoclonal mouse anti-Pax7 (Developmental Studies Hybridoma Bank), monoclonal mouse anti-Pax3 (Developmental Studies Hybridoma Bank) polyclonal rabbit anti-GFP (Invitrogen) and monoclonal mouse anti-MyHC (MF20) (Developmental Studies Hybridoma Bank).

    Techniques: Activity Assay, Expressing, Infection, Construct, Plasmid Preparation, Cell Culture, Immunostaining, Derivative Assay, Staining

    Quantitative analysis of the effects of Pax3 and Pax7 on myogenic gene expression. C2C12 were infected with control RV, Pax3 RV, Pax7 RV, Pax3DN RV or Pax7DN RV and analysed for mRNA (72 h later) and protein levels either 72 h or 1 week later. Semi-quantitative RT-PCR showed that constitutive Pax7 expression did not induce endogenous Pax3 mRNA expression, while Pax3DN did not down-regulate endogenous Pax7 mRNA (a – M: DNA ladder; NTC: no template control; amplicon sizes – Pax3 : 151 bp; Pax7 : 247 bp; Gapdh : 250 bp, representative experiments shown in duplicate). QPCR analysis of Myf5 , MyoD and myogenin expression levels 72 h post-infection demonstrated that constitutive Pax3 or Pax7 expression both increased Myf5 levels, whereas the dominant-negative versions had the opposite effects and decreased Myf5 expression (b). Pax3DN or Pax7DN also suppressed both MyoD and myogenin expression (b). Data show mean fold change from control RV-infected cells (mean±SEM, from duplicate measurements from 2 independent experiments n = 2+2). Hypothesis testing performed using REST analysis where an asterisk denotes significant difference at p

    Journal: PLoS ONE

    Article Title: Integrated Functions of Pax3 and Pax7 in the Regulation of Proliferation, Cell Size and Myogenic Differentiation

    doi: 10.1371/journal.pone.0004475

    Figure Lengend Snippet: Quantitative analysis of the effects of Pax3 and Pax7 on myogenic gene expression. C2C12 were infected with control RV, Pax3 RV, Pax7 RV, Pax3DN RV or Pax7DN RV and analysed for mRNA (72 h later) and protein levels either 72 h or 1 week later. Semi-quantitative RT-PCR showed that constitutive Pax7 expression did not induce endogenous Pax3 mRNA expression, while Pax3DN did not down-regulate endogenous Pax7 mRNA (a – M: DNA ladder; NTC: no template control; amplicon sizes – Pax3 : 151 bp; Pax7 : 247 bp; Gapdh : 250 bp, representative experiments shown in duplicate). QPCR analysis of Myf5 , MyoD and myogenin expression levels 72 h post-infection demonstrated that constitutive Pax3 or Pax7 expression both increased Myf5 levels, whereas the dominant-negative versions had the opposite effects and decreased Myf5 expression (b). Pax3DN or Pax7DN also suppressed both MyoD and myogenin expression (b). Data show mean fold change from control RV-infected cells (mean±SEM, from duplicate measurements from 2 independent experiments n = 2+2). Hypothesis testing performed using REST analysis where an asterisk denotes significant difference at p

    Article Snippet: Primary antibodies used were monoclonal rat anti-BrdU clone BU1/75 (Abcam), monoclonal mouse anti-myogenin clone F5D (Developmental Studies Hybridoma Bank), polyclonal rabbit anti-myogenin (Santa Cruz), monoclonal mouse anti-MyoD clone 5.8A (DakoCytomation), polyclonal rabbit anti-MyoD (Santa Cruz), monoclonal mouse anti-Pax7 (Developmental Studies Hybridoma Bank), monoclonal mouse anti-Pax3 (Developmental Studies Hybridoma Bank) polyclonal rabbit anti-GFP (Invitrogen) and monoclonal mouse anti-MyHC (MF20) (Developmental Studies Hybridoma Bank).

    Techniques: Expressing, Infection, Quantitative RT-PCR, Amplification, Real-time Polymerase Chain Reaction, Dominant Negative Mutation

    APC-null satellite cells lose their myogenic potential and undergo apoptosis upon activation. (A) Scheme of success ratio in generation of primary myoblasts culture from TM-treated control and APC SC-KO mice. (B) Pax7, MyoD, and Myogenin immunostaining on myogenic colonies obtained from single EDL fibers cultured on Matrigel for 7 d. (C) Quantification of the number of myogenic and nonmyogenic cells per colony obtained in B. P-value refers to the myogenic proportion. (D) Pax7 immunostaining on single EDL fibers isolated and fixed (0 h) or cultured in floating conditions for 30 or 48 h (broken lines indicate the underlying fiber). (E) Quantification of BrdU-incorporating satellite cells on EDL fibers after 24 h of culture. (F) Quantification of satellite cell doublets per fiber after 30 h of culture. (G) Quantification of total number of satellite cells per fiber after 0, 30, and 48 h of culture. (H) Pax7 and activated Caspase3 (act-Casp3) immunostaining on EDL single fibers after 30 h of culture. (I) Quantification of apoptotic satellite cells after 30 h of culture. (J) Quantification of the percentage of satellite cells on EDL fibers expressing Myogenin after 30 h of culture. (K) Schematic representation of the in vivo analysis of satellite cell proliferation and survival upon muscle injury. (L and M) Quantification of BrdU-incorporating satellite cells on regenerating EDL fibers (L) and of apoptotic cells by TUNEL staining on TA sections (M) 2 d after injury. Bars: (B) 100 µm; (D and H) 10 µm. Error bars indicate SEM. N.S., not significant (P > 0.05).

    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-null satellite cells lose their myogenic potential and undergo apoptosis upon activation. (A) Scheme of success ratio in generation of primary myoblasts culture from TM-treated control and APC SC-KO mice. (B) Pax7, MyoD, and Myogenin immunostaining on myogenic colonies obtained from single EDL fibers cultured on Matrigel for 7 d. (C) Quantification of the number of myogenic and nonmyogenic cells per colony obtained in B. P-value refers to the myogenic proportion. (D) Pax7 immunostaining on single EDL fibers isolated and fixed (0 h) or cultured in floating conditions for 30 or 48 h (broken lines indicate the underlying fiber). (E) Quantification of BrdU-incorporating satellite cells on EDL fibers after 24 h of culture. (F) Quantification of satellite cell doublets per fiber after 30 h of culture. (G) Quantification of total number of satellite cells per fiber after 0, 30, and 48 h of culture. (H) Pax7 and activated Caspase3 (act-Casp3) immunostaining on EDL single fibers after 30 h of culture. (I) Quantification of apoptotic satellite cells after 30 h of culture. (J) Quantification of the percentage of satellite cells on EDL fibers expressing Myogenin after 30 h of culture. (K) Schematic representation of the in vivo analysis of satellite cell proliferation and survival upon muscle injury. (L and M) Quantification of BrdU-incorporating satellite cells on regenerating EDL fibers (L) and of apoptotic cells by TUNEL staining on TA sections (M) 2 d after injury. Bars: (B) 100 µm; (D and H) 10 µm. Error bars indicate SEM. N.S., not significant (P > 0.05).

    Article Snippet: Primary antibodies are as follows: rabbit APC (Abcam), mouse BrdU (Dako), rabbit activated-Caspase3 (Cell Signaling Technology), rabbit activated β-catenin (Cell Signaling Technology), mouse MyoD (Dako), rabbit Myogenin (Santa Cruz Biotechnology, Inc.), mouse Pax7 (Developmental Studies Hybridoma Bank), and rabbit PCNA (Bethyl Laboratories, Inc.).

    Techniques: Activation Assay, Mouse Assay, Immunostaining, Cell Culture, Isolation, Activated Clotting Time Assay, Expressing, In Vivo, TUNEL Assay, Staining

    Proliferation and expression of myogenic regulatory factors MyoD and myogenin. (A) Proliferation was assessed using 5-ethynil-2'-deoxyuridine (EdU + ) following 6, 24, 48, and 72 hours in a growth medium. Myogenic regulatory factors (B) MyoD + and (C) Myogenin + nuclei were assessed following 24 and 72 hours in a differentiation medium. Representative images of (D) MyoD + and (E) Myogenin + nuclei at 24 hours. Scale bar = 50μm. * Significance p

    Journal: PLoS ONE

    Article Title: Preclinical characterization of the JAK/STAT inhibitor SGI-1252 on skeletal muscle function, morphology, and satellite cell content

    doi: 10.1371/journal.pone.0198611

    Figure Lengend Snippet: Proliferation and expression of myogenic regulatory factors MyoD and myogenin. (A) Proliferation was assessed using 5-ethynil-2'-deoxyuridine (EdU + ) following 6, 24, 48, and 72 hours in a growth medium. Myogenic regulatory factors (B) MyoD + and (C) Myogenin + nuclei were assessed following 24 and 72 hours in a differentiation medium. Representative images of (D) MyoD + and (E) Myogenin + nuclei at 24 hours. Scale bar = 50μm. * Significance p

    Article Snippet: Primary antibodies: MyoD—Monoclonal Mouse Anti-MyoD1 (Dako Company), Myogenin—Rabbit Polyclonal IgG Myogenin sc-576, Lot# G0313 (Santa Cruz Biotechnology), and Myosin Heavy Chain—MF 20-S mouse anti-myosin (Developmental Studies Hybridoma Bank).

    Techniques: Expressing

    Changes in the markers of satellite cells . Representative cross-sections of soleus muscle immunostained for M-cadherin, BrdU, myoD, and myogenin (green, arrows ), respectively. The two columns on the left are DIC images. The two columns on the right show dystrophin immunolabeling (red) to identify fiber profiles and nuclear staining with DAPI (blue). Scale bar = 20 μm.

    Journal: BMC Cell Biology

    Article Title: The effects of low frequency electrical stimulation on satellite cell activity in rat skeletal muscle during hindlimb suspension

    doi: 10.1186/1471-2121-11-87

    Figure Lengend Snippet: Changes in the markers of satellite cells . Representative cross-sections of soleus muscle immunostained for M-cadherin, BrdU, myoD, and myogenin (green, arrows ), respectively. The two columns on the left are DIC images. The two columns on the right show dystrophin immunolabeling (red) to identify fiber profiles and nuclear staining with DAPI (blue). Scale bar = 20 μm.

    Article Snippet: The sections were then incubated overnight at 4°C with rabbit polyclonal anti-M-cadherin (H-71, 1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-BrdU (BU-33, 1:50 dilution; Sigma), mouse monoclonal anti-myoD (5.8A, 1:50 dilution; Santa Cruz Biotechnology), or mouse monoclonal anti-myogenin (F12B, 1:100 dilution; Sigma) together with a goat polyclonal anti-dystrophin antibody (C-terminus, 1:50 dilution; Santa Cruz Biotechnology) for double staining.

    Techniques: Immunolabeling, Staining

    Quantification of satellite cells labeled positive in soleus muscles . Number of M-cadherin + cells or BrdU + , myoD + , and myogenin + nuclei per 100 fibers in cross sections of the soleus muscles. ** P

    Journal: BMC Cell Biology

    Article Title: The effects of low frequency electrical stimulation on satellite cell activity in rat skeletal muscle during hindlimb suspension

    doi: 10.1186/1471-2121-11-87

    Figure Lengend Snippet: Quantification of satellite cells labeled positive in soleus muscles . Number of M-cadherin + cells or BrdU + , myoD + , and myogenin + nuclei per 100 fibers in cross sections of the soleus muscles. ** P

    Article Snippet: The sections were then incubated overnight at 4°C with rabbit polyclonal anti-M-cadherin (H-71, 1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-BrdU (BU-33, 1:50 dilution; Sigma), mouse monoclonal anti-myoD (5.8A, 1:50 dilution; Santa Cruz Biotechnology), or mouse monoclonal anti-myogenin (F12B, 1:100 dilution; Sigma) together with a goat polyclonal anti-dystrophin antibody (C-terminus, 1:50 dilution; Santa Cruz Biotechnology) for double staining.

    Techniques: Labeling

    Myogenesis in adherent colonies derived from fiber-associated satellite cells. Satellite cell colonies were stained for c-met (red) and MyoD (green; A,C,E ) or marker myosin heavy chain (red; B,D,F ); nuclei were visualized with DAPI. Wild-type colonies form multinucleate myotubes, stain positively for MyoD ( A ), and express myosin heavy chain, indicating successful differentiation ( B ). Syndecan-3 -/- colonies form large, irregular syncytia, rarely express MyoD ( C ), and fail to express myosin heavy chain ( D ). Syndecan-4 -/- colonies completely fail to either form myotubes or to express MyoD ( E ) and only very rarely do single cells express myosin heavy chain ( F ). ( G ) Addition of exogenous heparin increased the number of cells per clone in mass cultures of wild-type and syndecan-3 -/- satellite cells but not syndecan-4 -/- satellite cells; bars represent standard deviations. Heparin also increased differentiation as measured by expression of nuclear MyoD (red) and myosin heavy chain (green) in syndecan-3 -/- cells (525 μg/mL shown; H ) and partially rescued the mutant phenotype in syndecan-4 -/- cells (525 μg/mL shown; I ).

    Journal: Genes & Development

    Article Title: Essential and separable roles for Syndecan-3 and Syndecan-4 in skeletal muscle development and regeneration

    doi: 10.1101/gad.1214204

    Figure Lengend Snippet: Myogenesis in adherent colonies derived from fiber-associated satellite cells. Satellite cell colonies were stained for c-met (red) and MyoD (green; A,C,E ) or marker myosin heavy chain (red; B,D,F ); nuclei were visualized with DAPI. Wild-type colonies form multinucleate myotubes, stain positively for MyoD ( A ), and express myosin heavy chain, indicating successful differentiation ( B ). Syndecan-3 -/- colonies form large, irregular syncytia, rarely express MyoD ( C ), and fail to express myosin heavy chain ( D ). Syndecan-4 -/- colonies completely fail to either form myotubes or to express MyoD ( E ) and only very rarely do single cells express myosin heavy chain ( F ). ( G ) Addition of exogenous heparin increased the number of cells per clone in mass cultures of wild-type and syndecan-3 -/- satellite cells but not syndecan-4 -/- satellite cells; bars represent standard deviations. Heparin also increased differentiation as measured by expression of nuclear MyoD (red) and myosin heavy chain (green) in syndecan-3 -/- cells (525 μg/mL shown; H ) and partially rescued the mutant phenotype in syndecan-4 -/- cells (525 μg/mL shown; I ).

    Article Snippet: Primary antibodies used were rabbit anti-laminin (Sigma) at 1:200, mouse anti-neurofilament (Sigma) at 1:100, rabbit anti-syndecan-3 at 1:100, chicken anti-syndecan-4 at 1:1500, rabbit anti-c-met (Santa Cruz) at 1:100, mouse anti-MyoD (Novocastra) at 1:10, and mouse anti-myosin heavy chain (MF20) neat.

    Techniques: Derivative Assay, Staining, Marker, Expressing, Mutagenesis

    Histological changes on day five after empty vector or BMP-2 gene transfer. a-e) Skeletal muscles on day five after empty vector transfer. a) Haematoxylin-eosin staining showing spindle-shaped and cytoplasm-enriched cells in the spaces between muscle fibres (arrows). b) CD68-positive cells (arrow). c) Pax7-positive cells (arrow). d) Myod1- positive cells (arrow). e) Myogenin-positive cells are detected around the cell migration area (arrows). f-k) Skeletal muscles on day five after BMP-2 gene transfer. f ) Haematoxylin-eosin staining showing typical muscle fibres. g) CD68-positive cells. h) BMP-2-positive cells are detected in the haematoxylin-positive population, but not in muscle fibres. i) Pax7-positive cells (arrow). j) M-cadherin-positive cells (arrows). k) Myogenin-positive cells (arrows) are detected between muscle fibres. Scale bars: a,b) 100 > m; c-k) 50 > m.

    Journal: European Journal of Histochemistry : EJH

    Article Title: Determination of cell fate in skeletal muscle following BMP gene transfer by in vivo electroporation

    doi: 10.4081/ejh.2017.2772

    Figure Lengend Snippet: Histological changes on day five after empty vector or BMP-2 gene transfer. a-e) Skeletal muscles on day five after empty vector transfer. a) Haematoxylin-eosin staining showing spindle-shaped and cytoplasm-enriched cells in the spaces between muscle fibres (arrows). b) CD68-positive cells (arrow). c) Pax7-positive cells (arrow). d) Myod1- positive cells (arrow). e) Myogenin-positive cells are detected around the cell migration area (arrows). f-k) Skeletal muscles on day five after BMP-2 gene transfer. f ) Haematoxylin-eosin staining showing typical muscle fibres. g) CD68-positive cells. h) BMP-2-positive cells are detected in the haematoxylin-positive population, but not in muscle fibres. i) Pax7-positive cells (arrow). j) M-cadherin-positive cells (arrows). k) Myogenin-positive cells (arrows) are detected between muscle fibres. Scale bars: a,b) 100 > m; c-k) 50 > m.

    Article Snippet: Anti-Pax7 rabbit polyclonal, anti-Myod1 mouse monoclonal, antimyogenin mouse monoclonal, anti-M-cadherin goat polyclonal, and anti-BMP-2 mouse monoclonal antibodies (all 1:100; Abcam, Cambridge, UK), and anti-CD68 mouse monoclonal antibody (1:50; Bio- Rad, Hercules, CA, USA) were incubated for 12 h at 4°C.

    Techniques: Plasmid Preparation, Staining, Migration

    Histological changes in skeletal muscles on day three after empty vector or BMP-2 gene transfer. a,b) Skeletal muscles on day three after empty vector transfer. a) Haematoxylin-eosin staining showing enlarged muscle fibres filled with lymphocyte-like cells (arrows). b) CD68-positive cells (arrows). c-f ) Histological changes on day three after BMP-2 gene transfer. c) Haematoxylin-eosin staining showing enlarged spaces between fibres (arrow). d) CD68-positive cells. e) BMP-2-positive cells are detected only in less damaged muscles (arrows). f ) Myod1-positive cells localised around the spaces between muscle fibres (arrows). Scale bars: 100 > m.

    Journal: European Journal of Histochemistry : EJH

    Article Title: Determination of cell fate in skeletal muscle following BMP gene transfer by in vivo electroporation

    doi: 10.4081/ejh.2017.2772

    Figure Lengend Snippet: Histological changes in skeletal muscles on day three after empty vector or BMP-2 gene transfer. a,b) Skeletal muscles on day three after empty vector transfer. a) Haematoxylin-eosin staining showing enlarged muscle fibres filled with lymphocyte-like cells (arrows). b) CD68-positive cells (arrows). c-f ) Histological changes on day three after BMP-2 gene transfer. c) Haematoxylin-eosin staining showing enlarged spaces between fibres (arrow). d) CD68-positive cells. e) BMP-2-positive cells are detected only in less damaged muscles (arrows). f ) Myod1-positive cells localised around the spaces between muscle fibres (arrows). Scale bars: 100 > m.

    Article Snippet: Anti-Pax7 rabbit polyclonal, anti-Myod1 mouse monoclonal, antimyogenin mouse monoclonal, anti-M-cadherin goat polyclonal, and anti-BMP-2 mouse monoclonal antibodies (all 1:100; Abcam, Cambridge, UK), and anti-CD68 mouse monoclonal antibody (1:50; Bio- Rad, Hercules, CA, USA) were incubated for 12 h at 4°C.

    Techniques: Plasmid Preparation, Staining

    dKO Cells Exhibit a Block in Muscle Differentiation (A–L) Expression of myogenin (MYOG) in injured muscle is dependent on MyoD or Myf5 . MYOG expression (arrowheads) was delayed or undetectable in Myf5-SA (F and H) and dKO (J and L) satellite cells, respectively. (M–T) Cultured dKO satellite cells fail to fuse or express MYOG or MyHC after 21 days in DM. (U–X) Co-culturing of equivalent numbers of wild-type and GFP+ dKO cells for 14 days in DM did not rescue fusion or differentiation capacity of dKO cells. Arrowheads identify representative fibers, which are GFP– and MyHC+. Scale bars represent 10 μm (A–L), 50 μm (M–T), or 100 μm (U–X).

    Journal: Stem Cell Reports

    Article Title: Loss of MyoD and Myf5 in Skeletal Muscle Stem Cells Results in Altered Myogenic Programming and Failed Regeneration

    doi: 10.1016/j.stemcr.2018.01.027

    Figure Lengend Snippet: dKO Cells Exhibit a Block in Muscle Differentiation (A–L) Expression of myogenin (MYOG) in injured muscle is dependent on MyoD or Myf5 . MYOG expression (arrowheads) was delayed or undetectable in Myf5-SA (F and H) and dKO (J and L) satellite cells, respectively. (M–T) Cultured dKO satellite cells fail to fuse or express MYOG or MyHC after 21 days in DM. (U–X) Co-culturing of equivalent numbers of wild-type and GFP+ dKO cells for 14 days in DM did not rescue fusion or differentiation capacity of dKO cells. Arrowheads identify representative fibers, which are GFP– and MyHC+. Scale bars represent 10 μm (A–L), 50 μm (M–T), or 100 μm (U–X).

    Article Snippet: The following primary antibodies were used: chicken anti-GFP (Abcam, 13970); rabbit anti-PLIN1 (Sigma-Aldrich, P1817); mouse anti-chicken PAX7 (Developmental Studies Hybridoma Bank [DSHB]); mouse anti-rat myogenin (mAb F5D; DSHB); rabbit anti-PCNA (Santa Cruz, FL-261); mouse anti-human MYOD (mAb 5.8A, BD Biosciences, 554130); rabbit anti-desmin (Sigma, P1873); rabbit anti-rat calcitonin receptor (Bio-Rad, AHP635); and mouse anti-chicken MyHC (mAb MF20; DSHB).

    Techniques: Blocking Assay, Expressing, Cell Culture

    Whole-Mount and Histological Analyses of Skeletal Muscle Regeneration Directed by Varying Gene Dosages of MyoD and Myf5 in Satellite Cells (A–D) Whole-mount images of uninjured and injured (11 dpi) TA muscles from MyoD-SA (A and B) and dKO (C and D) mice. Mice carried the Cre-dependent GFP reporter, R26 NG . Fluorescence images in (A) and (C) are shown in (B) and (D), respectively. GFP+ satellite cells in uninjured muscles are not visible at this magnification and exposure (B and D); top. The exposure times to capture the GFP images were 126 ms (B) and 892 ms (D). Scale bars represent 1 mm (A–D). (E–L) Histological analyses at 11 dpi (E–H) and 31 dpi (I–L). Sections were stained with H E (E–K) or oil red O (L). The same section is shown in (K and L). dKO muscle was filled with adipocytes (black asterisks) (K and L) and apparent fibrotic tissue (white asterisks) (K). Scale bars represent 50 μm. (M–O) Lineage-labeled dKO cells populated the injured TA muscle. The boxed area in (M) is magnified in (N). Occasional, minute fibers are evident in (O) (arrows). (P) Myf5-SA mice exhibited the typical architecture of regenerated muscle at 31 dpi. (Q–T) PCNA immunofluorescence of Myf5-SA and dKO satellite cells at 3 dpi. Yellow arrowheads indicate representative PCNA+ GFP+ cells. Scale bars represent 100 μm (M–P) and 25 μm (Q–T). .

    Journal: Stem Cell Reports

    Article Title: Loss of MyoD and Myf5 in Skeletal Muscle Stem Cells Results in Altered Myogenic Programming and Failed Regeneration

    doi: 10.1016/j.stemcr.2018.01.027

    Figure Lengend Snippet: Whole-Mount and Histological Analyses of Skeletal Muscle Regeneration Directed by Varying Gene Dosages of MyoD and Myf5 in Satellite Cells (A–D) Whole-mount images of uninjured and injured (11 dpi) TA muscles from MyoD-SA (A and B) and dKO (C and D) mice. Mice carried the Cre-dependent GFP reporter, R26 NG . Fluorescence images in (A) and (C) are shown in (B) and (D), respectively. GFP+ satellite cells in uninjured muscles are not visible at this magnification and exposure (B and D); top. The exposure times to capture the GFP images were 126 ms (B) and 892 ms (D). Scale bars represent 1 mm (A–D). (E–L) Histological analyses at 11 dpi (E–H) and 31 dpi (I–L). Sections were stained with H E (E–K) or oil red O (L). The same section is shown in (K and L). dKO muscle was filled with adipocytes (black asterisks) (K and L) and apparent fibrotic tissue (white asterisks) (K). Scale bars represent 50 μm. (M–O) Lineage-labeled dKO cells populated the injured TA muscle. The boxed area in (M) is magnified in (N). Occasional, minute fibers are evident in (O) (arrows). (P) Myf5-SA mice exhibited the typical architecture of regenerated muscle at 31 dpi. (Q–T) PCNA immunofluorescence of Myf5-SA and dKO satellite cells at 3 dpi. Yellow arrowheads indicate representative PCNA+ GFP+ cells. Scale bars represent 100 μm (M–P) and 25 μm (Q–T). .

    Article Snippet: The following primary antibodies were used: chicken anti-GFP (Abcam, 13970); rabbit anti-PLIN1 (Sigma-Aldrich, P1817); mouse anti-chicken PAX7 (Developmental Studies Hybridoma Bank [DSHB]); mouse anti-rat myogenin (mAb F5D; DSHB); rabbit anti-PCNA (Santa Cruz, FL-261); mouse anti-human MYOD (mAb 5.8A, BD Biosciences, 554130); rabbit anti-desmin (Sigma, P1873); rabbit anti-rat calcitonin receptor (Bio-Rad, AHP635); and mouse anti-chicken MyHC (mAb MF20; DSHB).

    Techniques: Mouse Assay, Fluorescence, Mass Spectrometry, Staining, Labeling, Immunofluorescence