mouse anti-pax7 Search Results


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  • 93
    Millipore mouse monoclonal anti pax7
    TRIM32 mutations seem to lead to premature senescence of myogenic cells. a Immunohistochemistry staining and quantification of <t>Pax7</t> + satellite cells in skeletal muscle from family A patients ( n = 2), family C patient ( n = 1), healthy controls ( n = 5) and disease controls (LGMD2B, LGMD1C, LGMD2N, NEM6) ( n = 4). Immunostaining for collagen type IV (red) to show the muscle fibers, Pax7 (green) to show the satellite cells and with Topro 3 staining for the nuclei (blue). Quantification of Pax7 + cells revealed a significant reduction in the number of satellite cells in TRIM32 V591M and TRIM32 C39LfsX17 muscles compared with controls. Data from 8 to 31 independent fields were analyzed per condition. Mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test. Scale bar, 50 μm. b Immunohistochemical staining of MHC-neo of skeletal muscle from family A patients, family B patients, healthy control and disease control (LGMD1B) revealed a large number of positive regenerating fibers in the disease control. In contrast, TRIM32 patients showed no positive cells (patients A/II.2, A/IV.3 and B/II.2) or, at most, few scattered positive cells (patients B/II.3 and C/II.2). Scale bar, 100 μm. c SEM images of myoblasts at 5 days growing in proliferation medium from AIV.3 and BII.3 patients, and healthy controls. TRIM32 V591M and TRIM32 N217S/F568del myoblasts were larger than control myoblasts. Higher magnification showed a reduction in the size of projections and number of filopodia of TRIM32 V591M and TRIM32 N217S/F568del myoblasts comparing to control myoblasts. Scale bars, 100 μm: lower magnification view; 2 μm: hyper magnification view. d Immunofluorescence staining and quantification of the percentage of SA-β-gal + cells in human myoblasts after 10 days growing in proliferation medium from family A patients ( n = 2), family B patients ( n = 2) and healthy controls ( n = 2). A higher increment of SA-β-gal + cells was observed in TRIM32 V591M and TRIM32 N217S/F568del myoblast cultures compared to controls, supporting a premature senescence in the muscles with TRIM32 altered function. Data from 8 independent fields were analyzed per condition. Mean ± SEM; One-way ANOVA with Tukey’s multiple comparisons test. Scale bar, 100 μm
    Mouse Monoclonal Anti Pax7, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Developmental Studies Hybridoma Bank mouse anti pax7
    Collapsed individual myofibers induce MPC proliferation. (A) Representative X-gal staining of intact and collapsed myofibers isolated from the EDL muscles of Myf5-nLacZ mice after 0 and 6 days of culture. Arrows indicate Myf5 + cells. (B) Representative photomicrographs of intact and collapsed myofibers cultured for 0 and 6 days and immunostained with a <t>Pax7-specific</t> antibody (red) and stained with fluorochrome-conjugated phalloidin and DAPI to reveal actin filaments (F-actin, green) and nuclei (blue), respectively. The arrows indicate MPCs (Pax7 + cells). (C) Scatter dot plot showing the number of Pax7 + cells per intact or collapsed myofiber after 6 days of culture. Myofibers from the EDL muscles of three adult WT mice were immunolabeled with a Pax7-specific antibody. Nuclei were stained with DAPI ( n = 0 to 43 myofibers). ** P = 0.0024 versus intact myofibers. (D) Percentage of the distribution of the number of Pax7 + cells per intact and collapsed myofiber after 6 days of culture. (E) Bar graphs representing the proportion of MPCs in various differentiation states for intact or collapsed myofibers after 0 and 6 days of culture. Following immunostaining of MPCs with Pax7 and MyoD, the differentiation states were defined in terms of combinations of positive staining: quiescent (Pax7 + MyoD − ), proliferative (Pax7 + MyoD + ), and differentiating (Pax7 − MyoD + ). The results are from three independent experiments. Results are expressed as means ± SEM. ** P
    Mouse Anti Pax7, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 1095 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems human mouse rat chicken pax7 antibody
    mDUX and myogenic regulators. qRT-PCR for mDUX and myogenic genes in iC2C12-mDUX cells evaluated at different times (A, using 500 ng/mL doxycycline) or doses (C, at 12 hours). Results are presented as fold difference compared to uninduced cells (0 ng/ml) except for the expression of mDUX in which 12 hours of induction was taken as the group for comparison. Error bars represent the STDEV. Induction with 8 ng/mL of doxycycline was sufficient for significant down-regulation of MyoD. (B) Immunofluorescence for detection of MyoD (red) in iC2C12-mDUX cells induced during the time course of 12 hours. Nuclei were stained with DAPI (blue). A notable decrease in the number of the positive-staining nuclei and the intensity of the staining was detected as early as 4 hours after induction. (C) Expression of mDUX, MyoD, and <t>Pax7</t> when mDUX is induced with various concentrations of doxycycline.
    Human Mouse Rat Chicken Pax7 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology mouse anti pax7
    Pharmacological role of CB1 in primary human muscle cells. a Representative blot for CB1 and <t>PAX7</t> protein expression in primary human satellite cells. The approximate molecular mass for each of these proteins (expressed in kDa) is shown on the right. b Bar graph showing the mRNA expression levels of MYOG and TNNT-1 in satellite cells exposed to DM for 5 days +/− rimonabant 1–3 µM. Each bar is the mean ± SEM of four separate determinations. c Morphological analysis of myotube formation in human primary satellite cells exposed to DM for 5 days in the presence of vehicle (DMSO, control) or rimonabant 1 µM. MyHC (red) and DAPI (blue). (Scale bar, 10 μm). The fusion index was calculated in both vehicle (DMSO)- and rimonabant-treated cells exposed to DM for 5 days. The asterisk denotes the P ≤ 0.05 vs. vehicle-treated cells. d Bar graphs showing the MYOG and TNNT-1 mRNA expression levels in primary human myoblasts isolated from each DMD donors (D1–D9) and induced to differentiate in presence of vehicle (DMSO) or rimonabant (1 µM). The quantification of transcripts was performed in quadruplicate by quantitative real-time PCR. The error bars correspond to the internal SEM. e The graph shows the differences in the expression levels of MYOG and TNNT-1 between vehicle- or rimonabant-treated myoblasts calculated by combining the DMD patient’s results together. Each bar is the mean ± SEM of the data from the nine patients, each of which was obtained from at least four separate determinations (see d ). * P ≤ 0.05 vs. vehicle group, determined by Student’s t test
    Mouse Anti Pax7, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Developmental Studies Hybridoma Bank mouse anti chicken pax7
    dKO Satellite Cells Express Markers of Myogenic Commitment after Muscle Injury (A–F) <t>PAX7</t> immunofluorescence of transverse cryosections of the TA muscle 31 dpi. PAX7+ cells (examples at yellow arrowheads) carrying a wild-type allele of MyoD (A and D) or Myf5 (B and E) were associated with regenerated fibers, identified by their central nucleation (examples at arrows). PAX7+ dKO cells were numerous at 31 dpi (C and F). Apparent PAX7 levels were reduced in dKO satellite cells. Exposure times were identical between genotypes. Scale bars represent 10 μm. (G–I) qRT-PCR analysis of myogenic gene expression of FACS-isolated satellite cells. ΔCt values were calculated using the average Ct values of the internal controls, Gapdh and EiF1a , which were highly consistent between genotypes within a given stage. Average Ct values were as follows: uninjured, 23.75; 3 dpi, 20.92; 12 dpi, 24.54. Each data point represents the average value of three biological samples and three technical replicates for each sample. Error bars represent standard deviations. p
    Mouse Anti Chicken Pax7, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam mouse anti pax7
    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 <t>Pax7,</t> 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
    Mouse Anti Pax7, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Developmental Studies Hybridoma Bank mouse igg1 anti pax7
    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 <t>Pax7,</t> 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
    Mouse Igg1 Anti Pax7, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 88/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Developmental Studies Hybridoma Bank mouse anti pax 7
    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 <t>Pax7,</t> 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
    Mouse Anti Pax 7, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    TRIM32 mutations seem to lead to premature senescence of myogenic cells. a Immunohistochemistry staining and quantification of Pax7 + satellite cells in skeletal muscle from family A patients ( n = 2), family C patient ( n = 1), healthy controls ( n = 5) and disease controls (LGMD2B, LGMD1C, LGMD2N, NEM6) ( n = 4). Immunostaining for collagen type IV (red) to show the muscle fibers, Pax7 (green) to show the satellite cells and with Topro 3 staining for the nuclei (blue). Quantification of Pax7 + cells revealed a significant reduction in the number of satellite cells in TRIM32 V591M and TRIM32 C39LfsX17 muscles compared with controls. Data from 8 to 31 independent fields were analyzed per condition. Mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test. Scale bar, 50 μm. b Immunohistochemical staining of MHC-neo of skeletal muscle from family A patients, family B patients, healthy control and disease control (LGMD1B) revealed a large number of positive regenerating fibers in the disease control. In contrast, TRIM32 patients showed no positive cells (patients A/II.2, A/IV.3 and B/II.2) or, at most, few scattered positive cells (patients B/II.3 and C/II.2). Scale bar, 100 μm. c SEM images of myoblasts at 5 days growing in proliferation medium from AIV.3 and BII.3 patients, and healthy controls. TRIM32 V591M and TRIM32 N217S/F568del myoblasts were larger than control myoblasts. Higher magnification showed a reduction in the size of projections and number of filopodia of TRIM32 V591M and TRIM32 N217S/F568del myoblasts comparing to control myoblasts. Scale bars, 100 μm: lower magnification view; 2 μm: hyper magnification view. d Immunofluorescence staining and quantification of the percentage of SA-β-gal + cells in human myoblasts after 10 days growing in proliferation medium from family A patients ( n = 2), family B patients ( n = 2) and healthy controls ( n = 2). A higher increment of SA-β-gal + cells was observed in TRIM32 V591M and TRIM32 N217S/F568del myoblast cultures compared to controls, supporting a premature senescence in the muscles with TRIM32 altered function. Data from 8 independent fields were analyzed per condition. Mean ± SEM; One-way ANOVA with Tukey’s multiple comparisons test. Scale bar, 100 μm

    Journal: Acta Neuropathologica Communications

    Article Title: Altered myogenesis and premature senescence underlie human TRIM32-related myopathy

    doi: 10.1186/s40478-019-0683-9

    Figure Lengend Snippet: TRIM32 mutations seem to lead to premature senescence of myogenic cells. a Immunohistochemistry staining and quantification of Pax7 + satellite cells in skeletal muscle from family A patients ( n = 2), family C patient ( n = 1), healthy controls ( n = 5) and disease controls (LGMD2B, LGMD1C, LGMD2N, NEM6) ( n = 4). Immunostaining for collagen type IV (red) to show the muscle fibers, Pax7 (green) to show the satellite cells and with Topro 3 staining for the nuclei (blue). Quantification of Pax7 + cells revealed a significant reduction in the number of satellite cells in TRIM32 V591M and TRIM32 C39LfsX17 muscles compared with controls. Data from 8 to 31 independent fields were analyzed per condition. Mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test. Scale bar, 50 μm. b Immunohistochemical staining of MHC-neo of skeletal muscle from family A patients, family B patients, healthy control and disease control (LGMD1B) revealed a large number of positive regenerating fibers in the disease control. In contrast, TRIM32 patients showed no positive cells (patients A/II.2, A/IV.3 and B/II.2) or, at most, few scattered positive cells (patients B/II.3 and C/II.2). Scale bar, 100 μm. c SEM images of myoblasts at 5 days growing in proliferation medium from AIV.3 and BII.3 patients, and healthy controls. TRIM32 V591M and TRIM32 N217S/F568del myoblasts were larger than control myoblasts. Higher magnification showed a reduction in the size of projections and number of filopodia of TRIM32 V591M and TRIM32 N217S/F568del myoblasts comparing to control myoblasts. Scale bars, 100 μm: lower magnification view; 2 μm: hyper magnification view. d Immunofluorescence staining and quantification of the percentage of SA-β-gal + cells in human myoblasts after 10 days growing in proliferation medium from family A patients ( n = 2), family B patients ( n = 2) and healthy controls ( n = 2). A higher increment of SA-β-gal + cells was observed in TRIM32 V591M and TRIM32 N217S/F568del myoblast cultures compared to controls, supporting a premature senescence in the muscles with TRIM32 altered function. Data from 8 independent fields were analyzed per condition. Mean ± SEM; One-way ANOVA with Tukey’s multiple comparisons test. Scale bar, 100 μm

    Article Snippet: Afterward, muscle samples were blocked with 2% non-fat milk + 0.3% Triton X-100 in PBS for 30 min and with 5% BSA + 0.5% Triton X-100 in PBS for 1 h. The following primary antibodies were used: mouse monoclonal anti-Pax7 (1:50; DHSB); mouse monoclonal anti-collagen VI (3C4) (1:1000; Chemicon) and incubated for 3 days at 4 °C.

    Techniques: Immunohistochemistry, Staining, Immunostaining, Immunofluorescence

    Collapsed individual myofibers induce MPC proliferation. (A) Representative X-gal staining of intact and collapsed myofibers isolated from the EDL muscles of Myf5-nLacZ mice after 0 and 6 days of culture. Arrows indicate Myf5 + cells. (B) Representative photomicrographs of intact and collapsed myofibers cultured for 0 and 6 days and immunostained with a Pax7-specific antibody (red) and stained with fluorochrome-conjugated phalloidin and DAPI to reveal actin filaments (F-actin, green) and nuclei (blue), respectively. The arrows indicate MPCs (Pax7 + cells). (C) Scatter dot plot showing the number of Pax7 + cells per intact or collapsed myofiber after 6 days of culture. Myofibers from the EDL muscles of three adult WT mice were immunolabeled with a Pax7-specific antibody. Nuclei were stained with DAPI ( n = 0 to 43 myofibers). ** P = 0.0024 versus intact myofibers. (D) Percentage of the distribution of the number of Pax7 + cells per intact and collapsed myofiber after 6 days of culture. (E) Bar graphs representing the proportion of MPCs in various differentiation states for intact or collapsed myofibers after 0 and 6 days of culture. Following immunostaining of MPCs with Pax7 and MyoD, the differentiation states were defined in terms of combinations of positive staining: quiescent (Pax7 + MyoD − ), proliferative (Pax7 + MyoD + ), and differentiating (Pax7 − MyoD + ). The results are from three independent experiments. Results are expressed as means ± SEM. ** P

    Journal: Skeletal Muscle

    Article Title: Increased microenvironment stiffness in damaged myofibers promotes myogenic progenitor cell proliferation

    doi: 10.1186/s13395-015-0030-1

    Figure Lengend Snippet: Collapsed individual myofibers induce MPC proliferation. (A) Representative X-gal staining of intact and collapsed myofibers isolated from the EDL muscles of Myf5-nLacZ mice after 0 and 6 days of culture. Arrows indicate Myf5 + cells. (B) Representative photomicrographs of intact and collapsed myofibers cultured for 0 and 6 days and immunostained with a Pax7-specific antibody (red) and stained with fluorochrome-conjugated phalloidin and DAPI to reveal actin filaments (F-actin, green) and nuclei (blue), respectively. The arrows indicate MPCs (Pax7 + cells). (C) Scatter dot plot showing the number of Pax7 + cells per intact or collapsed myofiber after 6 days of culture. Myofibers from the EDL muscles of three adult WT mice were immunolabeled with a Pax7-specific antibody. Nuclei were stained with DAPI ( n = 0 to 43 myofibers). ** P = 0.0024 versus intact myofibers. (D) Percentage of the distribution of the number of Pax7 + cells per intact and collapsed myofiber after 6 days of culture. (E) Bar graphs representing the proportion of MPCs in various differentiation states for intact or collapsed myofibers after 0 and 6 days of culture. Following immunostaining of MPCs with Pax7 and MyoD, the differentiation states were defined in terms of combinations of positive staining: quiescent (Pax7 + MyoD − ), proliferative (Pax7 + MyoD + ), and differentiating (Pax7 − MyoD + ). The results are from three independent experiments. Results are expressed as means ± SEM. ** P

    Article Snippet: The cells, myofibers, and tissue sections were incubated with mouse anti-Pax7 (1:2; DSHB, Iowa City, IA, USA), mouse anti-myogenin (1:3; DSHB), mouse anti-α-actinin (1:1,000; Sigma), rabbit anti-MyoD (1:200; C-20, Santa Cruz, CA, USA), rabbit anti-fibronectin (1:100; Millipore, Billerica, MA, USA), or rabbit anti-Ki67 (1:100; Abcam, Cambridge, UK) primary antibodies or with Alexa 488-conjugated phalloidin (1:1,000; Invitrogen, Canada).

    Techniques: Staining, Isolation, Mouse Assay, Cell Culture, Immunolabeling, Immunostaining

    Pax7 inhibits the development of tectofugal projections

    Journal:

    Article Title: Regulation of the development of tectal neurons and their projections by transcription factors Brn3a and Pax7

    doi: 10.1016/j.ydbio.2007.12.040

    Figure Lengend Snippet: Pax7 inhibits the development of tectofugal projections

    Article Snippet: Antibodies used were: rabbit anti-Brn3a , rabbit anti-GFP (Abcam), chicken anti-GFP (Avian Laboratories), mouse monoclonal anti-Pax7 (Developmental Studies Hybridoma Bank), goat anti-beta-galactosidase (Biogenesis), rabbit anti-intermediate neurofilament (Novus Biologicals).

    Techniques:

    Birthdating of Brn3a and Pax7 expressing tectal neurons

    Journal:

    Article Title: Regulation of the development of tectal neurons and their projections by transcription factors Brn3a and Pax7

    doi: 10.1016/j.ydbio.2007.12.040

    Figure Lengend Snippet: Birthdating of Brn3a and Pax7 expressing tectal neurons

    Article Snippet: Antibodies used were: rabbit anti-Brn3a , rabbit anti-GFP (Abcam), chicken anti-GFP (Avian Laboratories), mouse monoclonal anti-Pax7 (Developmental Studies Hybridoma Bank), goat anti-beta-galactosidase (Biogenesis), rabbit anti-intermediate neurofilament (Novus Biologicals).

    Techniques: Expressing

    Lamina-specific expression of Brn3a and Pax7 in the chick tectum

    Journal:

    Article Title: Regulation of the development of tectal neurons and their projections by transcription factors Brn3a and Pax7

    doi: 10.1016/j.ydbio.2007.12.040

    Figure Lengend Snippet: Lamina-specific expression of Brn3a and Pax7 in the chick tectum

    Article Snippet: Antibodies used were: rabbit anti-Brn3a , rabbit anti-GFP (Abcam), chicken anti-GFP (Avian Laboratories), mouse monoclonal anti-Pax7 (Developmental Studies Hybridoma Bank), goat anti-beta-galactosidase (Biogenesis), rabbit anti-intermediate neurofilament (Novus Biologicals).

    Techniques: Expressing

    Freshly sorted satellite cells express the satellite cell markers Pax7 and Syndecan −4

    Journal: Developmental biology

    Article Title: A cell-autonomous defect in skeletal muscle satellite cells expressing low levels of survival of motor neuron protein

    doi: 10.1016/j.ydbio.2012.05.037

    Figure Lengend Snippet: Freshly sorted satellite cells express the satellite cell markers Pax7 and Syndecan −4

    Article Snippet: Cells were fixed (4% paraformaldehyde, 10 min), permeabilized with 0.2% Triton X-100/PBS, blocked with 50:50 MOM Blocking (Vector Laboratories, Burlingame, CA) and 10% horse serum in PBS, and stained with chicken anti-syndecan-4 (1:400; Olwin laboratory, University of Colorado Boulder), mouse anti-Pax7 antibody (1:25; Developmental Studies Hybridoma Bank, Iowa City, IA), rabbit anti-MyoD antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-myogenin (1:400; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques:

    Six1 negatively regulates ERK signaling in SCs. (A) qRT-PCR analysis of Six1 , Ang1 , Tie2 , Etv4 , and Dusp6 expression in proliferating myoblasts. Six1 gene disruption or silencing increases Ang1 and Etv4 transcription levels and decreases Dusp6 expression. (B) qRT-PCR analysis of Six1 and Dusp6 expression in proliferating myoblasts. Six1 overexpression increases Dusp6 transcription. (C) Real-time PCR analysis of locus enrichment in ChIP from proliferating myoblasts. Six1 proteins are bound to the MEF3 element upstream of the Dusp6 gene. (D) Immunolocalization of Pax7 and Dusp6 proteins on 3-d cultured EDL myofibers. Proliferating Six1KO SCs do not express Dusp6. (E) Detection of phosphorylated ERK from control (gray) and Six1KO (fushia) myoblasts. pI values are plotted against signal intensities. Different ERK isoforms and the relative increases in signal intensity of Six1KO over control are indicated ( n = 3). Six1KO myoblasts present elevated ERK1 signaling ex vivo. (F) Immunolocalization of Pax7 and phosphorylated ERK1/2 proteins on 7-d regenerating EDL muscles. Six1KO SCs present elevated ERK signaling in vivo. (G) Immunolocalization of Pax7 and phosphorylated ERK1/2 proteins on 6-d cultured EDL myofibers. Six1KO SCs present elevated ERK signaling ex vivo. (H) EDL myofibers from control and Erk1 −/− animals. Immunostaining indicated that quiescent SCs express Pax7 + and have a correct sublaminar position in mutant muscles. Pax7 + sublaminar SCs were scored on EDL single fibers (left) and on TA cryosections (right). The SC pool is diminished in muscles from Erk1 −/− mice compared with controls. Error bars indicate standard deviations. *, P

    Journal: The Journal of Cell Biology

    Article Title: Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration

    doi: 10.1083/jcb.201201050

    Figure Lengend Snippet: Six1 negatively regulates ERK signaling in SCs. (A) qRT-PCR analysis of Six1 , Ang1 , Tie2 , Etv4 , and Dusp6 expression in proliferating myoblasts. Six1 gene disruption or silencing increases Ang1 and Etv4 transcription levels and decreases Dusp6 expression. (B) qRT-PCR analysis of Six1 and Dusp6 expression in proliferating myoblasts. Six1 overexpression increases Dusp6 transcription. (C) Real-time PCR analysis of locus enrichment in ChIP from proliferating myoblasts. Six1 proteins are bound to the MEF3 element upstream of the Dusp6 gene. (D) Immunolocalization of Pax7 and Dusp6 proteins on 3-d cultured EDL myofibers. Proliferating Six1KO SCs do not express Dusp6. (E) Detection of phosphorylated ERK from control (gray) and Six1KO (fushia) myoblasts. pI values are plotted against signal intensities. Different ERK isoforms and the relative increases in signal intensity of Six1KO over control are indicated ( n = 3). Six1KO myoblasts present elevated ERK1 signaling ex vivo. (F) Immunolocalization of Pax7 and phosphorylated ERK1/2 proteins on 7-d regenerating EDL muscles. Six1KO SCs present elevated ERK signaling in vivo. (G) Immunolocalization of Pax7 and phosphorylated ERK1/2 proteins on 6-d cultured EDL myofibers. Six1KO SCs present elevated ERK signaling ex vivo. (H) EDL myofibers from control and Erk1 −/− animals. Immunostaining indicated that quiescent SCs express Pax7 + and have a correct sublaminar position in mutant muscles. Pax7 + sublaminar SCs were scored on EDL single fibers (left) and on TA cryosections (right). The SC pool is diminished in muscles from Erk1 −/− mice compared with controls. Error bars indicate standard deviations. *, P

    Article Snippet: Primary antibodies used in this study were as follows: rat α7-Integrin (R & D Systems), rat CD34 (BD), goat Collagen Type I (SouthernBiotech), rabbit Desmin (Abcam), rabbit Dusp6 (Abcam), mouse Dystrophin (Novocastra), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), rabbit Myf5 (Santa Cruz Biotechnology, Inc.), rabbit MyoD (Santa Cruz Biotechnology, Inc.), mouse Myogenin (Dako), mouse MyHC embryonic (Vector Laboratories), mouse MyHC total (Developmental Studies Hybridoma Bank), mouse Pax7 (Developmental Studies Hybridoma Bank), rabbit Phospho-ERK1/2 (Cell Signaling Technology), rabbit IgG (Santa Cruz Biotechnology, Inc.), Rabbit Six1 (Sigma-Aldrich), rabbit Six4 (Antibodies Online), and chicken Syndecan4 (a gift from B. Olwin, University of Colorado, Boulder, CO).

    Techniques: Quantitative RT-PCR, Expressing, Over Expression, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Cell Culture, Ex Vivo, In Vivo, Immunostaining, Mutagenesis, Mouse Assay

    Dusp6 is required for restoring the SC pool during regeneration. TA and EDL muscles of control and Dusp6 −/− mice were injured by a single CTX injection and analyzed 30 d after the injury. (A) Single myofibers isolated from 30-d regenerated EDL muscles. Renewed Pax7 + SCs are located around host myofibers (arrows). (B) The SC pool is increased 2.4-fold on regenerated in Dusp6 −/− myofibers compared with control myofibers. (C) Cryosections of 30-d regenerated TA muscles. Immunolocalization of Pax7 and Laminin proteins allows visualization of sublaminar renewed SCs (arrows). (D) The SC pool is increased twofold within regenerated Dusp6 −/− muscles compared with control muscles. (E) Quantification of muscle fiber caliber in uninjured and regenerated TA muscles of Dusp6 −/− and control animals. No muscle defects were observed in mutant mice. Error bars indicate standard deviations. *, P

    Journal: The Journal of Cell Biology

    Article Title: Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration

    doi: 10.1083/jcb.201201050

    Figure Lengend Snippet: Dusp6 is required for restoring the SC pool during regeneration. TA and EDL muscles of control and Dusp6 −/− mice were injured by a single CTX injection and analyzed 30 d after the injury. (A) Single myofibers isolated from 30-d regenerated EDL muscles. Renewed Pax7 + SCs are located around host myofibers (arrows). (B) The SC pool is increased 2.4-fold on regenerated in Dusp6 −/− myofibers compared with control myofibers. (C) Cryosections of 30-d regenerated TA muscles. Immunolocalization of Pax7 and Laminin proteins allows visualization of sublaminar renewed SCs (arrows). (D) The SC pool is increased twofold within regenerated Dusp6 −/− muscles compared with control muscles. (E) Quantification of muscle fiber caliber in uninjured and regenerated TA muscles of Dusp6 −/− and control animals. No muscle defects were observed in mutant mice. Error bars indicate standard deviations. *, P

    Article Snippet: Primary antibodies used in this study were as follows: rat α7-Integrin (R & D Systems), rat CD34 (BD), goat Collagen Type I (SouthernBiotech), rabbit Desmin (Abcam), rabbit Dusp6 (Abcam), mouse Dystrophin (Novocastra), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), rabbit Myf5 (Santa Cruz Biotechnology, Inc.), rabbit MyoD (Santa Cruz Biotechnology, Inc.), mouse Myogenin (Dako), mouse MyHC embryonic (Vector Laboratories), mouse MyHC total (Developmental Studies Hybridoma Bank), mouse Pax7 (Developmental Studies Hybridoma Bank), rabbit Phospho-ERK1/2 (Cell Signaling Technology), rabbit IgG (Santa Cruz Biotechnology, Inc.), Rabbit Six1 (Sigma-Aldrich), rabbit Six4 (Antibodies Online), and chicken Syndecan4 (a gift from B. Olwin, University of Colorado, Boulder, CO).

    Techniques: Mouse Assay, Injection, Isolation, Mutagenesis

    Six1 gene disruption does not influence SC quiescence, activation, or proliferation. (A) Single myofibers isolated from EDL muscles of control ( Tg:Pax7-CreERT2::Six1 flox/+ ) and Six1KO (Tg:Pax7 CreERT2 /Six1 flox/flox ) mice 1 wk after TM injection. Six1 protein expression is lost in Six1KO SCs (arrows). (B) The majority of SCs from Six1KO EDL and TA muscles are negative for Six1 expression. (C) Quantification of quiescent sublaminar Pax7 + SCs per EDL myofibers isolated from control and Six1KO mice 6 wk after TM injection. Six1 loss does not perturb SC quiescence in vivo. (D) EDL myofibers from control and Six1KO animals were cultured for 2 d to visualize SC activation (Pax7 + /Ki67 + ). Six1 loss does not perturb SC activation ex vivo. (E) EDL myofibers from control and Six1KO animals were cultured for 3 d. SC descendants were immunolocalized for both Pax7 and Myogenin proteins. Six1 loss does not perturb SC proliferation ex vivo. (F) Primary myoblasts were isolated from control and Six1KO limb muscles. qRT-PCR analysis indicated expression of Six1 , Pax7 , and Six4 transcripts. Six1 gene disruption does not have an impact on Pax7 and Six4 expression levels. Error bars indicate standard deviations. *, P

    Journal: The Journal of Cell Biology

    Article Title: Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration

    doi: 10.1083/jcb.201201050

    Figure Lengend Snippet: Six1 gene disruption does not influence SC quiescence, activation, or proliferation. (A) Single myofibers isolated from EDL muscles of control ( Tg:Pax7-CreERT2::Six1 flox/+ ) and Six1KO (Tg:Pax7 CreERT2 /Six1 flox/flox ) mice 1 wk after TM injection. Six1 protein expression is lost in Six1KO SCs (arrows). (B) The majority of SCs from Six1KO EDL and TA muscles are negative for Six1 expression. (C) Quantification of quiescent sublaminar Pax7 + SCs per EDL myofibers isolated from control and Six1KO mice 6 wk after TM injection. Six1 loss does not perturb SC quiescence in vivo. (D) EDL myofibers from control and Six1KO animals were cultured for 2 d to visualize SC activation (Pax7 + /Ki67 + ). Six1 loss does not perturb SC activation ex vivo. (E) EDL myofibers from control and Six1KO animals were cultured for 3 d. SC descendants were immunolocalized for both Pax7 and Myogenin proteins. Six1 loss does not perturb SC proliferation ex vivo. (F) Primary myoblasts were isolated from control and Six1KO limb muscles. qRT-PCR analysis indicated expression of Six1 , Pax7 , and Six4 transcripts. Six1 gene disruption does not have an impact on Pax7 and Six4 expression levels. Error bars indicate standard deviations. *, P

    Article Snippet: Primary antibodies used in this study were as follows: rat α7-Integrin (R & D Systems), rat CD34 (BD), goat Collagen Type I (SouthernBiotech), rabbit Desmin (Abcam), rabbit Dusp6 (Abcam), mouse Dystrophin (Novocastra), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), rabbit Myf5 (Santa Cruz Biotechnology, Inc.), rabbit MyoD (Santa Cruz Biotechnology, Inc.), mouse Myogenin (Dako), mouse MyHC embryonic (Vector Laboratories), mouse MyHC total (Developmental Studies Hybridoma Bank), mouse Pax7 (Developmental Studies Hybridoma Bank), rabbit Phospho-ERK1/2 (Cell Signaling Technology), rabbit IgG (Santa Cruz Biotechnology, Inc.), Rabbit Six1 (Sigma-Aldrich), rabbit Six4 (Antibodies Online), and chicken Syndecan4 (a gift from B. Olwin, University of Colorado, Boulder, CO).

    Techniques: Activation Assay, Isolation, Mouse Assay, Injection, Expressing, In Vivo, Cell Culture, Ex Vivo, Quantitative RT-PCR

    Six1 gene disruption increases SCs self-renewal, but does not perturb the orientation of SC divisions. (A) FACS-sorted SCs were plated ex vivo and fixed after the first division. Typical doublets of sister SCs with Pax7 +/+ or Pax7 +/− gene signature are shown. (B) Six1KO SCs have higher self-renewal potential. (C) Immunolocalization of Pax7 and Ki67 proteins on myofibers separated from 4-d regenerating EDL muscles. (D) Quantification of SC division orientation. Six1 gene disruption does not have an impact on the rate of planar-to-perpendicular divisions. (E) EDL myofibers were separated from 4-d regenerating EDL muscles, and immunolocalized for Pax7 and Myf5 protein expression. Six1 gene disruption does not have an impact on the Myf5-negative satellite stem cell population. (F) FACS-sorted SCs separated on the basis of Myf5-Cre–driven reporter fluorescence and plated ex vivo. Immunolocalization of Pax7 and YFP proteins on YFP − (stem) and YFP + (committed) myoblasts. (G) qRT-PCR analysis indicated expression of Six1 transcripts by YFP + and YFP − SCs and myoblasts. Error bars indicate standard deviations. *, P

    Journal: The Journal of Cell Biology

    Article Title: Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration

    doi: 10.1083/jcb.201201050

    Figure Lengend Snippet: Six1 gene disruption increases SCs self-renewal, but does not perturb the orientation of SC divisions. (A) FACS-sorted SCs were plated ex vivo and fixed after the first division. Typical doublets of sister SCs with Pax7 +/+ or Pax7 +/− gene signature are shown. (B) Six1KO SCs have higher self-renewal potential. (C) Immunolocalization of Pax7 and Ki67 proteins on myofibers separated from 4-d regenerating EDL muscles. (D) Quantification of SC division orientation. Six1 gene disruption does not have an impact on the rate of planar-to-perpendicular divisions. (E) EDL myofibers were separated from 4-d regenerating EDL muscles, and immunolocalized for Pax7 and Myf5 protein expression. Six1 gene disruption does not have an impact on the Myf5-negative satellite stem cell population. (F) FACS-sorted SCs separated on the basis of Myf5-Cre–driven reporter fluorescence and plated ex vivo. Immunolocalization of Pax7 and YFP proteins on YFP − (stem) and YFP + (committed) myoblasts. (G) qRT-PCR analysis indicated expression of Six1 transcripts by YFP + and YFP − SCs and myoblasts. Error bars indicate standard deviations. *, P

    Article Snippet: Primary antibodies used in this study were as follows: rat α7-Integrin (R & D Systems), rat CD34 (BD), goat Collagen Type I (SouthernBiotech), rabbit Desmin (Abcam), rabbit Dusp6 (Abcam), mouse Dystrophin (Novocastra), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), rabbit Myf5 (Santa Cruz Biotechnology, Inc.), rabbit MyoD (Santa Cruz Biotechnology, Inc.), mouse Myogenin (Dako), mouse MyHC embryonic (Vector Laboratories), mouse MyHC total (Developmental Studies Hybridoma Bank), mouse Pax7 (Developmental Studies Hybridoma Bank), rabbit Phospho-ERK1/2 (Cell Signaling Technology), rabbit IgG (Santa Cruz Biotechnology, Inc.), Rabbit Six1 (Sigma-Aldrich), rabbit Six4 (Antibodies Online), and chicken Syndecan4 (a gift from B. Olwin, University of Colorado, Boulder, CO).

    Techniques: FACS, Ex Vivo, Expressing, Fluorescence, Quantitative RT-PCR

    Six1 activates MyoD and Myogenin expression by SCs in vivo. (A) qRT-PCR analysis of differentiating myogenic cells shows transient up-regulation of Six1 , MyoD , and Myogenin expressions during the first day after serum removal. (B) qRT-PCR analysis of differentiating myogenic cells shows that Six1 silencing decreases MyoD and Myogenin expression but not Myf5 expression ex vivo. (C) EDL single myofibers were cultured for 3 d, and immunolocalized for Pax7 and MyoD protein expression. Representative myogenic cell clusters are shown. (D) Percentage of SC descendants at the surface of cultured myofibers. Loss of Six1 decreases the proportion of committed cells (Pax7 − /MyoD + ) and increases the proportion of undifferentiated (Pax7 + /MyoD − ) cells in clusters. (E) Cryosections of 4-d regenerating TA muscles. Laminin staining shows basal lamina of myofibers. Immunolocalization of Myogenin proteins marks differentiating myonuclei. (F) Six1 gene disruption in SCs results in decreased Myogenin + nuclei numbers during muscle regeneration. (G) qRT-PCR analysis of Six1 , MyoD , and Myogenin transcripts levels by 4-d regenerating TA muscles. Six1 gene disruption decreases MyoD and Myogenin expression in vivo. (H) Schematic representations of Myogenin , MyoD , and Myf5 regulatory regions region. Shown are the localization and sequences of E-box (bHLH binding) and MEF3 (SIX binding) sites. (I) qRT-PCR analysis of locus enrichment in ChIP assays from differentiating myogenic cells. MyoD and Six1 proteins are bound to the MyoD and Myogenin upstream regulatory elements, but not on Myf5 enhancer. Error bars indicate standard deviations. *, P

    Journal: The Journal of Cell Biology

    Article Title: Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration

    doi: 10.1083/jcb.201201050

    Figure Lengend Snippet: Six1 activates MyoD and Myogenin expression by SCs in vivo. (A) qRT-PCR analysis of differentiating myogenic cells shows transient up-regulation of Six1 , MyoD , and Myogenin expressions during the first day after serum removal. (B) qRT-PCR analysis of differentiating myogenic cells shows that Six1 silencing decreases MyoD and Myogenin expression but not Myf5 expression ex vivo. (C) EDL single myofibers were cultured for 3 d, and immunolocalized for Pax7 and MyoD protein expression. Representative myogenic cell clusters are shown. (D) Percentage of SC descendants at the surface of cultured myofibers. Loss of Six1 decreases the proportion of committed cells (Pax7 − /MyoD + ) and increases the proportion of undifferentiated (Pax7 + /MyoD − ) cells in clusters. (E) Cryosections of 4-d regenerating TA muscles. Laminin staining shows basal lamina of myofibers. Immunolocalization of Myogenin proteins marks differentiating myonuclei. (F) Six1 gene disruption in SCs results in decreased Myogenin + nuclei numbers during muscle regeneration. (G) qRT-PCR analysis of Six1 , MyoD , and Myogenin transcripts levels by 4-d regenerating TA muscles. Six1 gene disruption decreases MyoD and Myogenin expression in vivo. (H) Schematic representations of Myogenin , MyoD , and Myf5 regulatory regions region. Shown are the localization and sequences of E-box (bHLH binding) and MEF3 (SIX binding) sites. (I) qRT-PCR analysis of locus enrichment in ChIP assays from differentiating myogenic cells. MyoD and Six1 proteins are bound to the MyoD and Myogenin upstream regulatory elements, but not on Myf5 enhancer. Error bars indicate standard deviations. *, P

    Article Snippet: Primary antibodies used in this study were as follows: rat α7-Integrin (R & D Systems), rat CD34 (BD), goat Collagen Type I (SouthernBiotech), rabbit Desmin (Abcam), rabbit Dusp6 (Abcam), mouse Dystrophin (Novocastra), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), rabbit Myf5 (Santa Cruz Biotechnology, Inc.), rabbit MyoD (Santa Cruz Biotechnology, Inc.), mouse Myogenin (Dako), mouse MyHC embryonic (Vector Laboratories), mouse MyHC total (Developmental Studies Hybridoma Bank), mouse Pax7 (Developmental Studies Hybridoma Bank), rabbit Phospho-ERK1/2 (Cell Signaling Technology), rabbit IgG (Santa Cruz Biotechnology, Inc.), Rabbit Six1 (Sigma-Aldrich), rabbit Six4 (Antibodies Online), and chicken Syndecan4 (a gift from B. Olwin, University of Colorado, Boulder, CO).

    Techniques: Expressing, In Vivo, Quantitative RT-PCR, Ex Vivo, Cell Culture, Staining, Binding Assay, Chromatin Immunoprecipitation

    Six1 gene disruption perturbs myogenic differentiation of SC descendants ex vivo. 1 wk after TM treatment, EDL myofibers from control and Six1KO mice were plated on Matrigel, and cultures were analyzed after 6 and 9 d of culture ex vivo. (A) Myogenic cells grown for 6 d were immunolocalized for Desmin (myoblast marker) and MyHC (differentiation marker) proteins. (B) Six1KO cells exhibit limited differentiation potential ex vivo compared with control cells. (C) Myogenic cells grown for 9 d were immunolocalized for Six1, Pax7 (undifferentiated state marker), and MyHC (differentiated state marker) proteins. (D) Six1KO cells fuse less efficiently and form smaller myotubes compared with control cells. (E) qRT-PCR analysis indicated expression of Six1 , CalcR , Pax7 ( SC markers ), and Myh1 , Myh4 (differentiation markers) transcripts by differentiated myogenic cells. (F) Six1KO cell cultures generate more Pax7 + cells compared with control cells. (G) SC-derived myogenic cells grown for 9 d were immunolocalized for Pax7 and both MyoD and Ki67 proteins. (H) Six1KO cells generate more “reserve” cells (Pax7 + /MyoD − /Ki67 − ; arrows) compared with control cells. Error bars indicate standard deviations. *, P

    Journal: The Journal of Cell Biology

    Article Title: Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration

    doi: 10.1083/jcb.201201050

    Figure Lengend Snippet: Six1 gene disruption perturbs myogenic differentiation of SC descendants ex vivo. 1 wk after TM treatment, EDL myofibers from control and Six1KO mice were plated on Matrigel, and cultures were analyzed after 6 and 9 d of culture ex vivo. (A) Myogenic cells grown for 6 d were immunolocalized for Desmin (myoblast marker) and MyHC (differentiation marker) proteins. (B) Six1KO cells exhibit limited differentiation potential ex vivo compared with control cells. (C) Myogenic cells grown for 9 d were immunolocalized for Six1, Pax7 (undifferentiated state marker), and MyHC (differentiated state marker) proteins. (D) Six1KO cells fuse less efficiently and form smaller myotubes compared with control cells. (E) qRT-PCR analysis indicated expression of Six1 , CalcR , Pax7 ( SC markers ), and Myh1 , Myh4 (differentiation markers) transcripts by differentiated myogenic cells. (F) Six1KO cell cultures generate more Pax7 + cells compared with control cells. (G) SC-derived myogenic cells grown for 9 d were immunolocalized for Pax7 and both MyoD and Ki67 proteins. (H) Six1KO cells generate more “reserve” cells (Pax7 + /MyoD − /Ki67 − ; arrows) compared with control cells. Error bars indicate standard deviations. *, P

    Article Snippet: Primary antibodies used in this study were as follows: rat α7-Integrin (R & D Systems), rat CD34 (BD), goat Collagen Type I (SouthernBiotech), rabbit Desmin (Abcam), rabbit Dusp6 (Abcam), mouse Dystrophin (Novocastra), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), rabbit Myf5 (Santa Cruz Biotechnology, Inc.), rabbit MyoD (Santa Cruz Biotechnology, Inc.), mouse Myogenin (Dako), mouse MyHC embryonic (Vector Laboratories), mouse MyHC total (Developmental Studies Hybridoma Bank), mouse Pax7 (Developmental Studies Hybridoma Bank), rabbit Phospho-ERK1/2 (Cell Signaling Technology), rabbit IgG (Santa Cruz Biotechnology, Inc.), Rabbit Six1 (Sigma-Aldrich), rabbit Six4 (Antibodies Online), and chicken Syndecan4 (a gift from B. Olwin, University of Colorado, Boulder, CO).

    Techniques: Ex Vivo, Mouse Assay, Marker, Quantitative RT-PCR, Expressing, Derivative Assay

    Six1 expression by SCs is necessary for proper skeletal muscle regeneration. (A) 3 d after TM treatment, TA muscles of control and Six1KO mice were injured by a single CTX injection and analyzed at various times during the regeneration process. (B) Cryosections of 4-d regenerating TA muscles. Immunolocalization of MyHC emb proteins marks the newly formed myofibers. (C) Regenerating myofibers of Six1KO animals are smaller compared with controls. (D) Regenerating myofibers of Six1KO animals contain fewer nuclei compared with controls. (E) Cryosections of 7-d regenerating TA muscles. Laminin staining shows basal lamina of myofibers. Strong MyHC emb staining marks a population of small, delayed myofibers. (F) 7-d regenerating muscles of Six1KO animals exhibit a significant proportion of lagged myofibers compared with controls. (G) 7-d regenerating control and Six1KO muscles do not contain significantly different amounts of Pax7 + cells. (H) Cryosections of regenerated TA muscles 14 d after CTX injection. Laminin staining shows basal lamina of myofibers. Note the abnormal accumulation of matrix in Six1KO muscles. (I) Quantification of muscle fiber caliber in 14-d regenerated TA muscles. Regenerated Six1KO muscles contain smaller fibers compared with regenerated control muscles. (J) Cryosections of regenerated TA muscles 14 d after CTX injection. Dystrophin staining shows myofibers sarcolemma. Shown is the percentage of Six1 + myonuclei. (K) Regeneration of the muscle tissue results in a higher number of nuclei per myofiber on cross sections in controls but not in Six1KO animals. Error bars indicate standard deviations. *, P

    Journal: The Journal of Cell Biology

    Article Title: Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration

    doi: 10.1083/jcb.201201050

    Figure Lengend Snippet: Six1 expression by SCs is necessary for proper skeletal muscle regeneration. (A) 3 d after TM treatment, TA muscles of control and Six1KO mice were injured by a single CTX injection and analyzed at various times during the regeneration process. (B) Cryosections of 4-d regenerating TA muscles. Immunolocalization of MyHC emb proteins marks the newly formed myofibers. (C) Regenerating myofibers of Six1KO animals are smaller compared with controls. (D) Regenerating myofibers of Six1KO animals contain fewer nuclei compared with controls. (E) Cryosections of 7-d regenerating TA muscles. Laminin staining shows basal lamina of myofibers. Strong MyHC emb staining marks a population of small, delayed myofibers. (F) 7-d regenerating muscles of Six1KO animals exhibit a significant proportion of lagged myofibers compared with controls. (G) 7-d regenerating control and Six1KO muscles do not contain significantly different amounts of Pax7 + cells. (H) Cryosections of regenerated TA muscles 14 d after CTX injection. Laminin staining shows basal lamina of myofibers. Note the abnormal accumulation of matrix in Six1KO muscles. (I) Quantification of muscle fiber caliber in 14-d regenerated TA muscles. Regenerated Six1KO muscles contain smaller fibers compared with regenerated control muscles. (J) Cryosections of regenerated TA muscles 14 d after CTX injection. Dystrophin staining shows myofibers sarcolemma. Shown is the percentage of Six1 + myonuclei. (K) Regeneration of the muscle tissue results in a higher number of nuclei per myofiber on cross sections in controls but not in Six1KO animals. Error bars indicate standard deviations. *, P

    Article Snippet: Primary antibodies used in this study were as follows: rat α7-Integrin (R & D Systems), rat CD34 (BD), goat Collagen Type I (SouthernBiotech), rabbit Desmin (Abcam), rabbit Dusp6 (Abcam), mouse Dystrophin (Novocastra), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), rabbit Myf5 (Santa Cruz Biotechnology, Inc.), rabbit MyoD (Santa Cruz Biotechnology, Inc.), mouse Myogenin (Dako), mouse MyHC embryonic (Vector Laboratories), mouse MyHC total (Developmental Studies Hybridoma Bank), mouse Pax7 (Developmental Studies Hybridoma Bank), rabbit Phospho-ERK1/2 (Cell Signaling Technology), rabbit IgG (Santa Cruz Biotechnology, Inc.), Rabbit Six1 (Sigma-Aldrich), rabbit Six4 (Antibodies Online), and chicken Syndecan4 (a gift from B. Olwin, University of Colorado, Boulder, CO).

    Techniques: Expressing, Mouse Assay, Injection, Staining

    Satellite cells express Six1. (A) qRT-PCR analysis indicated expression of SIX family transcripts by freshly FACS-sorted SCs (Satellites), myogenic cells cultured in growth medium (Myoblasts), or induced to differentiate by serum removal for 3 d (Myotubes). Error bars indicate standard deviations. (B) Single myofibers isolated from EDL muscles of C57BL/6 mice. Myofibers were cultured in floating conditions and immunolocalized for Six1 and Pax7 or Myogenin proteins at different times after isolation. All quiescent, dividing, or differentiating SCs expressed Six1. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration

    doi: 10.1083/jcb.201201050

    Figure Lengend Snippet: Satellite cells express Six1. (A) qRT-PCR analysis indicated expression of SIX family transcripts by freshly FACS-sorted SCs (Satellites), myogenic cells cultured in growth medium (Myoblasts), or induced to differentiate by serum removal for 3 d (Myotubes). Error bars indicate standard deviations. (B) Single myofibers isolated from EDL muscles of C57BL/6 mice. Myofibers were cultured in floating conditions and immunolocalized for Six1 and Pax7 or Myogenin proteins at different times after isolation. All quiescent, dividing, or differentiating SCs expressed Six1. Bars, 10 µm.

    Article Snippet: Primary antibodies used in this study were as follows: rat α7-Integrin (R & D Systems), rat CD34 (BD), goat Collagen Type I (SouthernBiotech), rabbit Desmin (Abcam), rabbit Dusp6 (Abcam), mouse Dystrophin (Novocastra), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), rabbit Myf5 (Santa Cruz Biotechnology, Inc.), rabbit MyoD (Santa Cruz Biotechnology, Inc.), mouse Myogenin (Dako), mouse MyHC embryonic (Vector Laboratories), mouse MyHC total (Developmental Studies Hybridoma Bank), mouse Pax7 (Developmental Studies Hybridoma Bank), rabbit Phospho-ERK1/2 (Cell Signaling Technology), rabbit IgG (Santa Cruz Biotechnology, Inc.), Rabbit Six1 (Sigma-Aldrich), rabbit Six4 (Antibodies Online), and chicken Syndecan4 (a gift from B. Olwin, University of Colorado, Boulder, CO).

    Techniques: Quantitative RT-PCR, Expressing, FACS, Cell Culture, Isolation, Mouse Assay

    Six1 limits SC self-renewal in vivo. (A) 3 d after TM treatment, TA muscles of control and Six1KO mice were injured by a single CTX injection and analyzed 30 d after the injury. (B) Single myofibers isolated from 30-d regenerated EDL muscles of control and Six1KO animals. Renewed Pax7 + SCs are located in sublaminar position around host myofibers in both control and Six1KO muscles. (C) Six1 is expressed by centrally located myonuclei and renewed SCs in control myofibers but not in Six1KO myofibers. Six1KO myofibers contain a higher number of renewed SCs (arrows). (D) Cryosections of 30-d regenerated TA muscles. Immunolocalization of Pax7 proteins mark quiescent SCs (arrows). (E) The SC pool is increased 2.4-fold in regenerated Six1KO TA muscles. (F) TA muscles of non-TM treated control and Six1KO mice were injured by a single CTX injection. Mice were then subjected to TM administration between 7 and 11 d after injury. Muscles were analyzed 30 d after the injury. (G) Cryosections of 30-d regenerated TA muscles. Immunolocalization of Pax7 proteins mark quiescent SCs (arrows). (H) Although the size of regenerated myofibers of control and Six1KO animals are similar, the SC pool is increased 2.1-fold in regenerated Six1KO TA muscles when TM was administrated after myogenesis has occurred. Error bars indicate standard deviations. *, P

    Journal: The Journal of Cell Biology

    Article Title: Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration

    doi: 10.1083/jcb.201201050

    Figure Lengend Snippet: Six1 limits SC self-renewal in vivo. (A) 3 d after TM treatment, TA muscles of control and Six1KO mice were injured by a single CTX injection and analyzed 30 d after the injury. (B) Single myofibers isolated from 30-d regenerated EDL muscles of control and Six1KO animals. Renewed Pax7 + SCs are located in sublaminar position around host myofibers in both control and Six1KO muscles. (C) Six1 is expressed by centrally located myonuclei and renewed SCs in control myofibers but not in Six1KO myofibers. Six1KO myofibers contain a higher number of renewed SCs (arrows). (D) Cryosections of 30-d regenerated TA muscles. Immunolocalization of Pax7 proteins mark quiescent SCs (arrows). (E) The SC pool is increased 2.4-fold in regenerated Six1KO TA muscles. (F) TA muscles of non-TM treated control and Six1KO mice were injured by a single CTX injection. Mice were then subjected to TM administration between 7 and 11 d after injury. Muscles were analyzed 30 d after the injury. (G) Cryosections of 30-d regenerated TA muscles. Immunolocalization of Pax7 proteins mark quiescent SCs (arrows). (H) Although the size of regenerated myofibers of control and Six1KO animals are similar, the SC pool is increased 2.1-fold in regenerated Six1KO TA muscles when TM was administrated after myogenesis has occurred. Error bars indicate standard deviations. *, P

    Article Snippet: Primary antibodies used in this study were as follows: rat α7-Integrin (R & D Systems), rat CD34 (BD), goat Collagen Type I (SouthernBiotech), rabbit Desmin (Abcam), rabbit Dusp6 (Abcam), mouse Dystrophin (Novocastra), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), rabbit Myf5 (Santa Cruz Biotechnology, Inc.), rabbit MyoD (Santa Cruz Biotechnology, Inc.), mouse Myogenin (Dako), mouse MyHC embryonic (Vector Laboratories), mouse MyHC total (Developmental Studies Hybridoma Bank), mouse Pax7 (Developmental Studies Hybridoma Bank), rabbit Phospho-ERK1/2 (Cell Signaling Technology), rabbit IgG (Santa Cruz Biotechnology, Inc.), Rabbit Six1 (Sigma-Aldrich), rabbit Six4 (Antibodies Online), and chicken Syndecan4 (a gift from B. Olwin, University of Colorado, Boulder, CO).

    Techniques: In Vivo, Mouse Assay, Injection, Isolation

    DUX4 and PAX7 are expressed in distinct cell types during myogenic differentiation. Representative images of DUX4 positive cells from D40 of the differentiation protocol stained with antibodies to both PAX7 and DUX4. a hESC-FSHD, b hiPSC-mosaic1-short, c hiPSC-mosaic2-short, and d hiPSC-mosaic1-long showing PAX7 positive nuclei but no DUX4 signal. Bar graph quantifying the e percentage of DUX4 positive nuclei and f percentage of PAX7 positive nuclei for the given number of DAPI-stained nuclei

    Journal: Skeletal Muscle

    Article Title: Expression patterns of FSHD-causing DUX4 and myogenic transcription factors PAX3 and PAX7 are spatially distinct in differentiating human stem cell cultures

    doi: 10.1186/s13395-017-0130-1

    Figure Lengend Snippet: DUX4 and PAX7 are expressed in distinct cell types during myogenic differentiation. Representative images of DUX4 positive cells from D40 of the differentiation protocol stained with antibodies to both PAX7 and DUX4. a hESC-FSHD, b hiPSC-mosaic1-short, c hiPSC-mosaic2-short, and d hiPSC-mosaic1-long showing PAX7 positive nuclei but no DUX4 signal. Bar graph quantifying the e percentage of DUX4 positive nuclei and f percentage of PAX7 positive nuclei for the given number of DAPI-stained nuclei

    Article Snippet: The primary antibodies used in this study were anti-PAX3 mouse monoclonal antibody (1:200 dilution, DSHB), anti-PAX7 mouse monoclonal antibody (1:200 dilution, DSHB), anti-Myogenin mouse monoclonal antibody (1:250 dilution, DSHB), anti-Titin mouse monoclonal antibody (1:200 dilution, DSHB), and anti-DUX4 E5-5 rabbit polyclonal antibody (1:1000 dilution, Abcam).

    Techniques: Staining

    RNA expression profiles of myogenic regulators PAX3 and PAX7 and DUX4 expression in cultures of human ES cells and iPS cell clones during myogenic differentiation. RNA was isolated at five time points (D0, D7, D21, D30, and D40) representing early, middle, and late stages of myogenesis. Quantitative PCR of PAX3, PAX7, and DUX4 RNA transcripts was performed at each time point, and values normalized to GAPDH RNA levels in the same preparations. Oligonucleotide primers designed to amplify the 3′ end of the DUX4 gene from the terminal D4Z4 unit on chromosome 4 were used to quantify DUX4 transcripts. Undetected RNA transcripts are marked with an arrow

    Journal: Skeletal Muscle

    Article Title: Expression patterns of FSHD-causing DUX4 and myogenic transcription factors PAX3 and PAX7 are spatially distinct in differentiating human stem cell cultures

    doi: 10.1186/s13395-017-0130-1

    Figure Lengend Snippet: RNA expression profiles of myogenic regulators PAX3 and PAX7 and DUX4 expression in cultures of human ES cells and iPS cell clones during myogenic differentiation. RNA was isolated at five time points (D0, D7, D21, D30, and D40) representing early, middle, and late stages of myogenesis. Quantitative PCR of PAX3, PAX7, and DUX4 RNA transcripts was performed at each time point, and values normalized to GAPDH RNA levels in the same preparations. Oligonucleotide primers designed to amplify the 3′ end of the DUX4 gene from the terminal D4Z4 unit on chromosome 4 were used to quantify DUX4 transcripts. Undetected RNA transcripts are marked with an arrow

    Article Snippet: The primary antibodies used in this study were anti-PAX3 mouse monoclonal antibody (1:200 dilution, DSHB), anti-PAX7 mouse monoclonal antibody (1:200 dilution, DSHB), anti-Myogenin mouse monoclonal antibody (1:250 dilution, DSHB), anti-Titin mouse monoclonal antibody (1:200 dilution, DSHB), and anti-DUX4 E5-5 rabbit polyclonal antibody (1:1000 dilution, Abcam).

    Techniques: RNA Expression, Expressing, Clone Assay, Isolation, Real-time Polymerase Chain Reaction

    Immunohistochemical analysis of Pax7 (brown nuclei) with methyl green counter staining (green nuclei). Original magnifications: ×200 ( A ); Immunoblotting of Pax7 ( B ). The graph represents the relative band density to β-tubulin. Data is shown as mean ± SD (** p

    Journal: International Journal of Molecular Sciences

    Article Title: Therapeutic Effect of Losartan, an Angiotensin II Type 1 Receptor Antagonist, on CCl4-Induced Skeletal Muscle Injury

    doi: 10.3390/ijms17020227

    Figure Lengend Snippet: Immunohistochemical analysis of Pax7 (brown nuclei) with methyl green counter staining (green nuclei). Original magnifications: ×200 ( A ); Immunoblotting of Pax7 ( B ). The graph represents the relative band density to β-tubulin. Data is shown as mean ± SD (** p

    Article Snippet: The sections were immunostained with a primary antibody: monoclonal mouse anti-dystrophin antibody (diluted to 1:10) (Novocastra Laboratories, Newcastle Ltd., Newcastle, UK), polyclonal rabbit anti-p-Smd2/3 antibody (diluted to 1:800) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal mouse anti-Pax7 antibody (diluted to 1:1000) (Developmental Studies Hybridoma Bank, Tokyo, Japan), monoclonal mouse anti-MyoD antibody (diluted to 1:100) (Santa Cruz Biotechnology, Inc.), or monoclonal mouse anti-myogenin antibody (diluted to 1:200) (Santa Cruz Biotechnology, Inc.).

    Techniques: Immunohistochemistry, Staining

    Immunocytochemistry of Pax3 and Pax7 expression in myogenic precursor cells, early-stage myoblasts, late-stage myoblasts, and differentiating myotubes at days 1–2 ( a , b ; i , j ), days 3–4 ( c , d ; k , l ), days 6–7 ( e , f; m , n ), and

    Journal: In vitro cellular & developmental biology. Animal

    Article Title: In vitro indeterminate teleost myogenesis appears to be dependent on Pax3

    doi: 10.1007/s11626-013-9616-2

    Figure Lengend Snippet: Immunocytochemistry of Pax3 and Pax7 expression in myogenic precursor cells, early-stage myoblasts, late-stage myoblasts, and differentiating myotubes at days 1–2 ( a , b ; i , j ), days 3–4 ( c , d ; k , l ), days 6–7 ( e , f; m , n ), and

    Article Snippet: Monoclonal mouse anti-Pax3 (ascites, 1:40 dilution) developed by C. P. Ordahl and monoclonal mouse anti-Pax7 (ascites, 1:40 dilution) developed by A. Kawakami were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.

    Techniques: Immunocytochemistry, Expressing

    Immunocytochemistry of Pax7 and MyoD1 expression in myogenic precursor cells cultured for 2.5 h ( a , b; e , f ) and 5 h ( c , d; g , h ) post-isolation. Anti-Pax7 detected by Texas Red fluorophore and anti-MyoD1 detected by FITC fluorophore. Total nuclei detected

    Journal: In vitro cellular & developmental biology. Animal

    Article Title: In vitro indeterminate teleost myogenesis appears to be dependent on Pax3

    doi: 10.1007/s11626-013-9616-2

    Figure Lengend Snippet: Immunocytochemistry of Pax7 and MyoD1 expression in myogenic precursor cells cultured for 2.5 h ( a , b; e , f ) and 5 h ( c , d; g , h ) post-isolation. Anti-Pax7 detected by Texas Red fluorophore and anti-MyoD1 detected by FITC fluorophore. Total nuclei detected

    Article Snippet: Monoclonal mouse anti-Pax3 (ascites, 1:40 dilution) developed by C. P. Ordahl and monoclonal mouse anti-Pax7 (ascites, 1:40 dilution) developed by A. Kawakami were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.

    Techniques: Immunocytochemistry, Expressing, Cell Culture, Isolation

    Summary of proposed roles of characterized SC subpopulations in myogenesis. In this study, two distinct SC subpopulations (P40/50 and P50/70) were isolated from muscle of 4-day-old piglets. Freshly isolated cells of the P40/50 subpopulation highly expressed Myf5 and MyoG and constituted a fast-proliferating phenotype. During in vitro cultivation, the P40/50 cells show a constantly high oxidative capacity, an increased differentiation potential, and fusion rates higher than the P50/70 cells. From these results, we assume that these cells by being a source of new myonuclei are the main contributors to hypertrophic growth of existing myofibers ( a ). Freshly isolated P50/70 cells showed considerably slower proliferation and expressed high amounts of markers for terminal differentiation ( Desmin, eMyH ). This leads us to suppose that some of the P50/70 cells are involved in tertiary fiber formation occurring with the highest intensity during the first week of pig postnatal muscle development ( b ). During culture, at least some of the P50/70 cells pass through a reversible period of low mitochondrial activity; this is associated with the appearance of higher numbers of Pax7 + cells and reduction of the differentiation potential/fusion rate compared with P40/50 cells whereas the proliferation rate equalized. Based on these results, we hypothesize that a subpopulation of P50/70 is withdrawn from differentiation to form and maintain a pool of slowly cycling, more immature precursor/reserve cells ( c ) at existing muscle fibers ( d ) gradual transformation to quiescent adult SC) that also can give rise to fast-proliferating, more committed (P40/50) cells ( e ) to prevent their exhaustion during intensive growth.

    Journal: Scientific Reports

    Article Title: Molecular and functional heterogeneity of early postnatal porcine satellite cell populations is associated with bioenergetic profile

    doi: 10.1038/srep45052

    Figure Lengend Snippet: Summary of proposed roles of characterized SC subpopulations in myogenesis. In this study, two distinct SC subpopulations (P40/50 and P50/70) were isolated from muscle of 4-day-old piglets. Freshly isolated cells of the P40/50 subpopulation highly expressed Myf5 and MyoG and constituted a fast-proliferating phenotype. During in vitro cultivation, the P40/50 cells show a constantly high oxidative capacity, an increased differentiation potential, and fusion rates higher than the P50/70 cells. From these results, we assume that these cells by being a source of new myonuclei are the main contributors to hypertrophic growth of existing myofibers ( a ). Freshly isolated P50/70 cells showed considerably slower proliferation and expressed high amounts of markers for terminal differentiation ( Desmin, eMyH ). This leads us to suppose that some of the P50/70 cells are involved in tertiary fiber formation occurring with the highest intensity during the first week of pig postnatal muscle development ( b ). During culture, at least some of the P50/70 cells pass through a reversible period of low mitochondrial activity; this is associated with the appearance of higher numbers of Pax7 + cells and reduction of the differentiation potential/fusion rate compared with P40/50 cells whereas the proliferation rate equalized. Based on these results, we hypothesize that a subpopulation of P50/70 is withdrawn from differentiation to form and maintain a pool of slowly cycling, more immature precursor/reserve cells ( c ) at existing muscle fibers ( d ) gradual transformation to quiescent adult SC) that also can give rise to fast-proliferating, more committed (P40/50) cells ( e ) to prevent their exhaustion during intensive growth.

    Article Snippet: Incubation with primary antibody mouse anti-Pax7 (Developmental Studies Hybridoma Bank, 1:50), mouse anti-Myogenin (abcam, 1:50), mouse anti-Desmin (DAKO, 1:80) or mouse anti-Myosin (skeletal, fast; Sigma Aldrich, 1:400) was performed overnight.

    Techniques: Isolation, In Vitro, Activity Assay, Transformation Assay

    Gene expression analysis of freshly isolated P40/50 and P50/70 cells. Gene expression analysis of the myogenic marker genes Pax7, Myf5, MyoD, Desmin, MyoG , and embryonic Myosin (eMyH ) of freshly isolated cells from LD muscle (n = 6). Quantitative real-time PCR shows the higher expression of transcription factors Myf5 and MyoG in P40/50 cells. In contrast, Desmin and eMyH are significantly upregulated in P50/70 cells. ΔΔCT values of each sample are presented as Box-Whisker plots with the maximum 1.5 of the interquartile range (Q 1 –Q 3 ), and the resulting outliers are included as circles. For statistical analysis, Students t-test ( Pax7, MyoD, Desmin, MyoG, eMyH ) or Mann-Whitney Rank Sum Test ( Myf5 ) was performed, **p ≤ 0.01, ***p ≤ 0.001.

    Journal: Scientific Reports

    Article Title: Molecular and functional heterogeneity of early postnatal porcine satellite cell populations is associated with bioenergetic profile

    doi: 10.1038/srep45052

    Figure Lengend Snippet: Gene expression analysis of freshly isolated P40/50 and P50/70 cells. Gene expression analysis of the myogenic marker genes Pax7, Myf5, MyoD, Desmin, MyoG , and embryonic Myosin (eMyH ) of freshly isolated cells from LD muscle (n = 6). Quantitative real-time PCR shows the higher expression of transcription factors Myf5 and MyoG in P40/50 cells. In contrast, Desmin and eMyH are significantly upregulated in P50/70 cells. ΔΔCT values of each sample are presented as Box-Whisker plots with the maximum 1.5 of the interquartile range (Q 1 –Q 3 ), and the resulting outliers are included as circles. For statistical analysis, Students t-test ( Pax7, MyoD, Desmin, MyoG, eMyH ) or Mann-Whitney Rank Sum Test ( Myf5 ) was performed, **p ≤ 0.01, ***p ≤ 0.001.

    Article Snippet: Incubation with primary antibody mouse anti-Pax7 (Developmental Studies Hybridoma Bank, 1:50), mouse anti-Myogenin (abcam, 1:50), mouse anti-Desmin (DAKO, 1:80) or mouse anti-Myosin (skeletal, fast; Sigma Aldrich, 1:400) was performed overnight.

    Techniques: Expressing, Isolation, Marker, Real-time Polymerase Chain Reaction, Whisker Assay, MANN-WHITNEY

    P40/50 and P50/70 differ in myogenic marker expression after prolonged cultivation. ( a ) P40/50 and P50/70 cells were passaged at day 4 and cultured in growth medium for another 5 days. Subsequently, immunofluorescence staining was performed to visualize Pax7; cell nuclei were stained with DAPI. Pax7 was localized in the nuclei, and a higher proportion of nuclei was positive for the transcription factor in P50/70 cells, in contrast to P40/50 cells. Percentages of positive cells of each sample (n = 5) are presented as Box-Whisker plots with the maximum 1.5 of the interquartile range (Q 1 –Q 3 ), and the resulting outliers are included as circles. For statistical analysis, Mann-Whitney Rank Sum Test was performed, *p ≤ 0.05. ( b ) Flow cytometric analysis of proliferating SC from SM or LD muscle after 8 days of cultivation. P40/50 cells show a significantly higher proportion of cells positive for Pax7. Percentages of positive cells of each sample are presented as Box-Whisker plots with the maximum 1.5 of the interquartile range (Q 1 –Q 3 ), and the resulting outliers are included as circles. For statistical analysis, Students t-test (Desmin, MyoG, MyHC) or Mann-Whitney Rank Sum Test (Pax7) was performed, *p ≤ 0.05, n = 4 (MyoG, MyHC), 5 (Pax7) and 9 (Desmin).

    Journal: Scientific Reports

    Article Title: Molecular and functional heterogeneity of early postnatal porcine satellite cell populations is associated with bioenergetic profile

    doi: 10.1038/srep45052

    Figure Lengend Snippet: P40/50 and P50/70 differ in myogenic marker expression after prolonged cultivation. ( a ) P40/50 and P50/70 cells were passaged at day 4 and cultured in growth medium for another 5 days. Subsequently, immunofluorescence staining was performed to visualize Pax7; cell nuclei were stained with DAPI. Pax7 was localized in the nuclei, and a higher proportion of nuclei was positive for the transcription factor in P50/70 cells, in contrast to P40/50 cells. Percentages of positive cells of each sample (n = 5) are presented as Box-Whisker plots with the maximum 1.5 of the interquartile range (Q 1 –Q 3 ), and the resulting outliers are included as circles. For statistical analysis, Mann-Whitney Rank Sum Test was performed, *p ≤ 0.05. ( b ) Flow cytometric analysis of proliferating SC from SM or LD muscle after 8 days of cultivation. P40/50 cells show a significantly higher proportion of cells positive for Pax7. Percentages of positive cells of each sample are presented as Box-Whisker plots with the maximum 1.5 of the interquartile range (Q 1 –Q 3 ), and the resulting outliers are included as circles. For statistical analysis, Students t-test (Desmin, MyoG, MyHC) or Mann-Whitney Rank Sum Test (Pax7) was performed, *p ≤ 0.05, n = 4 (MyoG, MyHC), 5 (Pax7) and 9 (Desmin).

    Article Snippet: Incubation with primary antibody mouse anti-Pax7 (Developmental Studies Hybridoma Bank, 1:50), mouse anti-Myogenin (abcam, 1:50), mouse anti-Desmin (DAKO, 1:80) or mouse anti-Myosin (skeletal, fast; Sigma Aldrich, 1:400) was performed overnight.

    Techniques: Marker, Expressing, Cell Culture, Immunofluorescence, Staining, Whisker Assay, MANN-WHITNEY, Flow Cytometry

    mDUX and myogenic regulators. qRT-PCR for mDUX and myogenic genes in iC2C12-mDUX cells evaluated at different times (A, using 500 ng/mL doxycycline) or doses (C, at 12 hours). Results are presented as fold difference compared to uninduced cells (0 ng/ml) except for the expression of mDUX in which 12 hours of induction was taken as the group for comparison. Error bars represent the STDEV. Induction with 8 ng/mL of doxycycline was sufficient for significant down-regulation of MyoD. (B) Immunofluorescence for detection of MyoD (red) in iC2C12-mDUX cells induced during the time course of 12 hours. Nuclei were stained with DAPI (blue). A notable decrease in the number of the positive-staining nuclei and the intensity of the staining was detected as early as 4 hours after induction. (C) Expression of mDUX, MyoD, and Pax7 when mDUX is induced with various concentrations of doxycycline.

    Journal: PLoS ONE

    Article Title: Biphasic Myopathic Phenotype of Mouse DUX, an ORF within Conserved FSHD-Related Repeats

    doi: 10.1371/journal.pone.0007003

    Figure Lengend Snippet: mDUX and myogenic regulators. qRT-PCR for mDUX and myogenic genes in iC2C12-mDUX cells evaluated at different times (A, using 500 ng/mL doxycycline) or doses (C, at 12 hours). Results are presented as fold difference compared to uninduced cells (0 ng/ml) except for the expression of mDUX in which 12 hours of induction was taken as the group for comparison. Error bars represent the STDEV. Induction with 8 ng/mL of doxycycline was sufficient for significant down-regulation of MyoD. (B) Immunofluorescence for detection of MyoD (red) in iC2C12-mDUX cells induced during the time course of 12 hours. Nuclei were stained with DAPI (blue). A notable decrease in the number of the positive-staining nuclei and the intensity of the staining was detected as early as 4 hours after induction. (C) Expression of mDUX, MyoD, and Pax7 when mDUX is induced with various concentrations of doxycycline.

    Article Snippet: The cells were incubated with the primary antibodies (mouse monoclonal anti mouse MyoD (1∶250, BD Biosciences), mouse monoclonal anti-Pax3 and anti-Pax7 (1∶250, R & D Systems), mouse anti-myosin heavy chain (1∶20, Developmental Studies Hybridoma Bank, U Iowa), polyclonal chicken anti-GFP antibody (1∶500, AbCam) diluted in PBS/3% BSA at 4°C overnight.

    Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining

    Pax3 and Pax7 compete with mDUX. (A) FACS analysis of iC2C12-mDUX cells transduced with MSCV retroviral constructs caring GFP, Pax3-ires-GFP or Pax7-ires-GFP. Almost all of the cells at the time of the experiment stably express GFP (x-axis). (B) Immunofluorescence for Pax3 or Pax7 (red) and GFP (green) reveals that Pax3 and Pax7 are expressed in GFP + cells. Cell number is decreased in induced samples due to toxicity of mDUX. (C) ATP assay for determination of cell viability in iC2C12-mDUX cells transduced with MSCV-ires-GFP, MSCV-Pax3-ires-GFP or MSCV-Pax7-ires-GFP. Cells were induced with various concentrations of doxycycline for 24 and 48 hours. Pax3- and Pax7-expressing cells are largely resistant to the toxicity of mDUX induced by 32 ng/mL dox even after 48 hours of induction. (D) qRT-PCR analyses for MyoD and Myf5 in the cells shown in (C), induced for 18 hours. Expression of MyoD and Myf5 is strongly repressed at 32 ng/mL induction in the control cells, but not the Pax3 or Pax7 expressing cells.

    Journal: PLoS ONE

    Article Title: Biphasic Myopathic Phenotype of Mouse DUX, an ORF within Conserved FSHD-Related Repeats

    doi: 10.1371/journal.pone.0007003

    Figure Lengend Snippet: Pax3 and Pax7 compete with mDUX. (A) FACS analysis of iC2C12-mDUX cells transduced with MSCV retroviral constructs caring GFP, Pax3-ires-GFP or Pax7-ires-GFP. Almost all of the cells at the time of the experiment stably express GFP (x-axis). (B) Immunofluorescence for Pax3 or Pax7 (red) and GFP (green) reveals that Pax3 and Pax7 are expressed in GFP + cells. Cell number is decreased in induced samples due to toxicity of mDUX. (C) ATP assay for determination of cell viability in iC2C12-mDUX cells transduced with MSCV-ires-GFP, MSCV-Pax3-ires-GFP or MSCV-Pax7-ires-GFP. Cells were induced with various concentrations of doxycycline for 24 and 48 hours. Pax3- and Pax7-expressing cells are largely resistant to the toxicity of mDUX induced by 32 ng/mL dox even after 48 hours of induction. (D) qRT-PCR analyses for MyoD and Myf5 in the cells shown in (C), induced for 18 hours. Expression of MyoD and Myf5 is strongly repressed at 32 ng/mL induction in the control cells, but not the Pax3 or Pax7 expressing cells.

    Article Snippet: The cells were incubated with the primary antibodies (mouse monoclonal anti mouse MyoD (1∶250, BD Biosciences), mouse monoclonal anti-Pax3 and anti-Pax7 (1∶250, R & D Systems), mouse anti-myosin heavy chain (1∶20, Developmental Studies Hybridoma Bank, U Iowa), polyclonal chicken anti-GFP antibody (1∶500, AbCam) diluted in PBS/3% BSA at 4°C overnight.

    Techniques: FACS, Transduction, Construct, Stable Transfection, Immunofluorescence, ATP Assay, Expressing, Quantitative RT-PCR

    Skeletal muscle development from human embryonic stem ( hES ) and human induced pluripotent stem ( hiPS ) cells by the EB culture method. (A) PAX3- and PAX7-positive nuclei emerged in the proximal area of the embryoid body (EB)-outgrowth cells derived from hES KhES1 cells. (B) Simultaneous derivation of neural and cardiac cells in the EB-outgrowth cells derived from hES KhES1 cells. Upper: TUJ1-positive neural cells observed on day 7+28. Lower: Neural cells (outlined arrowheads) and colonies of beating cardiomyocytes (white arrowhead) appeared on day 7+28. (C) Skeletal myosin-positive myofibers in the EB-outgrowth cells derived from human embryonic stem (hES) KhES1 cells detected on day 7+42. (D) Sequential analysis of undifferentiated and skeletal myogenesis-related gene expression by semi-quantitative RT-PCR. (E) Skeletal myosin-positive fibers from human induced pluripotent stem (hiPS) cells. Four hiPS cell-lines were used. hiPS 201B6 on day 7+105, hiPS 201B7 on day 7+105, hiPS 253G1 on day 7+77, and hiPS 253G4 on day 7+56. (F) Sequential analysis of undifferentiated and skeletal myogenesis-related gene expression by semi-quantitative RT-PCR. In (A-C) and (E), antibodies were visualized using Cy3 (red). Nuclei were counterstained with DAPI (blue). Scale bars = 100 µm.

    Journal: PLoS ONE

    Article Title: Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells

    doi: 10.1371/journal.pone.0051638

    Figure Lengend Snippet: Skeletal muscle development from human embryonic stem ( hES ) and human induced pluripotent stem ( hiPS ) cells by the EB culture method. (A) PAX3- and PAX7-positive nuclei emerged in the proximal area of the embryoid body (EB)-outgrowth cells derived from hES KhES1 cells. (B) Simultaneous derivation of neural and cardiac cells in the EB-outgrowth cells derived from hES KhES1 cells. Upper: TUJ1-positive neural cells observed on day 7+28. Lower: Neural cells (outlined arrowheads) and colonies of beating cardiomyocytes (white arrowhead) appeared on day 7+28. (C) Skeletal myosin-positive myofibers in the EB-outgrowth cells derived from human embryonic stem (hES) KhES1 cells detected on day 7+42. (D) Sequential analysis of undifferentiated and skeletal myogenesis-related gene expression by semi-quantitative RT-PCR. (E) Skeletal myosin-positive fibers from human induced pluripotent stem (hiPS) cells. Four hiPS cell-lines were used. hiPS 201B6 on day 7+105, hiPS 201B7 on day 7+105, hiPS 253G1 on day 7+77, and hiPS 253G4 on day 7+56. (F) Sequential analysis of undifferentiated and skeletal myogenesis-related gene expression by semi-quantitative RT-PCR. In (A-C) and (E), antibodies were visualized using Cy3 (red). Nuclei were counterstained with DAPI (blue). Scale bars = 100 µm.

    Article Snippet: The primary antibodies used in this study were as follows: mouse anti-Pax3 (R & D Systems, Minneapolis, MN, USA), mouse anti-Pax7 (R & D Systems), rabbit anti-Myf5 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-MyoD1, mouse anti-myogenin (Dako, Carpinteria, CA, USA), mouse anti-fast twitch myosin heavy chain (MYH2) (MY32; Zymed Laboratories, San Francisco, CA, USA), rabbit anti-skeletal myosin (Sigma), mouse anti-dystrophin (MANDRA1; Sigma), rabbit anti-laminin (Dako), mouse anti-human merosin (Laminin alpha 2), mouse anti-human lamin A/C (Novocastra Laboratories, Newcastle-upon-Tyne, UK), mouse anti-beta-tubulin III (TUJ1) (Sigma), and rabbit anti-glial fibrillary acidic protein antibody (GFAP) (Sigma).

    Techniques: Derivative Assay, Expressing, Quantitative RT-PCR

    Engraftment of myogenic progenitors in damaged muscles of immunodeficient mice. (A) Human nuclei labeled with human-specific lamin A/C localized mainly inside muscle fibers surrounded by laminin. (B) Muscle reconstruction by transplanted human cells was demonstrated by the detection of human-specific laminin-alpha 2. (C) The proportion of myofibers containing human nuclei at 4, 12, and 24 weeks after transplantation. (D) The proportion of myofibers containing human nuclei in reinjured (3+1 weeks) and in non-reinjured mice (4 weeks) at 4 weeks after transplantation. In C and D, data are presented as the mean ± standard deviation. (E) Distribution of the transplanted cells at 24 weeks after transplantation. Typical central nuclei of human origin were observed (outlined arrowheads). Some human cells located within the lamina rara beneath the basal lamina, indicating engraftment of the transplanted cells into a satellite cell compartment (white arrowhead). (F) Triple-staining for human Lamin A/C, PAX7, and pan-Laminin clearly demonstrated the existence of PAX7-positive human nuclei indicating the transplanted cells engrafted as satellite cells (white arrowhead). Human lamin A/C-negative host satellite cells were also detected (outlined arrowhead). Laminin was stained by a polyclonal antibody that recognizes both human and murine laminin, and was subsequently visualized with fluorescein isothiocyanate (FITC) (Green); human lamin A/C and human-specific laminin, with Cy3 (red). Nuclei were counterstained with DAPI (blue). Scale bars = (A) 100 µm, (B) and (E) 50 µm.

    Journal: PLoS ONE

    Article Title: Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells

    doi: 10.1371/journal.pone.0051638

    Figure Lengend Snippet: Engraftment of myogenic progenitors in damaged muscles of immunodeficient mice. (A) Human nuclei labeled with human-specific lamin A/C localized mainly inside muscle fibers surrounded by laminin. (B) Muscle reconstruction by transplanted human cells was demonstrated by the detection of human-specific laminin-alpha 2. (C) The proportion of myofibers containing human nuclei at 4, 12, and 24 weeks after transplantation. (D) The proportion of myofibers containing human nuclei in reinjured (3+1 weeks) and in non-reinjured mice (4 weeks) at 4 weeks after transplantation. In C and D, data are presented as the mean ± standard deviation. (E) Distribution of the transplanted cells at 24 weeks after transplantation. Typical central nuclei of human origin were observed (outlined arrowheads). Some human cells located within the lamina rara beneath the basal lamina, indicating engraftment of the transplanted cells into a satellite cell compartment (white arrowhead). (F) Triple-staining for human Lamin A/C, PAX7, and pan-Laminin clearly demonstrated the existence of PAX7-positive human nuclei indicating the transplanted cells engrafted as satellite cells (white arrowhead). Human lamin A/C-negative host satellite cells were also detected (outlined arrowhead). Laminin was stained by a polyclonal antibody that recognizes both human and murine laminin, and was subsequently visualized with fluorescein isothiocyanate (FITC) (Green); human lamin A/C and human-specific laminin, with Cy3 (red). Nuclei were counterstained with DAPI (blue). Scale bars = (A) 100 µm, (B) and (E) 50 µm.

    Article Snippet: The primary antibodies used in this study were as follows: mouse anti-Pax3 (R & D Systems, Minneapolis, MN, USA), mouse anti-Pax7 (R & D Systems), rabbit anti-Myf5 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-MyoD1, mouse anti-myogenin (Dako, Carpinteria, CA, USA), mouse anti-fast twitch myosin heavy chain (MYH2) (MY32; Zymed Laboratories, San Francisco, CA, USA), rabbit anti-skeletal myosin (Sigma), mouse anti-dystrophin (MANDRA1; Sigma), rabbit anti-laminin (Dako), mouse anti-human merosin (Laminin alpha 2), mouse anti-human lamin A/C (Novocastra Laboratories, Newcastle-upon-Tyne, UK), mouse anti-beta-tubulin III (TUJ1) (Sigma), and rabbit anti-glial fibrillary acidic protein antibody (GFAP) (Sigma).

    Techniques: Mouse Assay, Labeling, Transplantation Assay, Standard Deviation, Staining

    Engraftment of Transplanted GMSCs at the Wound Site and Expression of Myogenic Transcriptional Factors. (A) H E staining results of cross section of specimen taken after GMSC/SIS-ECM construction implantation at 2 weeks. Blue circle indicates the area of myomucosal regeneration. Muscle regeneration was not identified until 2 weeks in all groups (original magnification ×4). The distribution of PKH26 prelabeled GMSCs at the circled regeneration area was observed under a fluorescence microscope (original magnification ×10). The nuclei were counterstained with DAPI. Coexpression of MyoD ( green ) (B) , Myf5 ( green ) (C) , PAX7 ( green ) (D) , and PKH26 ( red ) in GMSCs at local defect area after 2 weeks transplantation was determined by immunostaining and observed under a fluorescence microscope. The nuclei were counterstained with DAPI (original magnification ×20).

    Journal: Tissue Engineering. Part A

    Article Title: A Gingiva-Derived Mesenchymal Stem Cell-Laden Porcine Small Intestinal Submucosa Extracellular Matrix Construct Promotes Myomucosal Regeneration of the Tongue

    doi: 10.1089/ten.tea.2016.0342

    Figure Lengend Snippet: Engraftment of Transplanted GMSCs at the Wound Site and Expression of Myogenic Transcriptional Factors. (A) H E staining results of cross section of specimen taken after GMSC/SIS-ECM construction implantation at 2 weeks. Blue circle indicates the area of myomucosal regeneration. Muscle regeneration was not identified until 2 weeks in all groups (original magnification ×4). The distribution of PKH26 prelabeled GMSCs at the circled regeneration area was observed under a fluorescence microscope (original magnification ×10). The nuclei were counterstained with DAPI. Coexpression of MyoD ( green ) (B) , Myf5 ( green ) (C) , PAX7 ( green ) (D) , and PKH26 ( red ) in GMSCs at local defect area after 2 weeks transplantation was determined by immunostaining and observed under a fluorescence microscope. The nuclei were counterstained with DAPI (original magnification ×20).

    Article Snippet: At day 14 post-transplantation of the PKH26-labeled GMSC/SIS-ECM construct, frozen sections were harvested and immunostained with antibodies for MyoD, Myf5, and PAX7, three myogenic transcription factors., Using immunofluorescent studies, we showed that PKH26 labeled GMSCs transplanted with SIS-ECM scaffold engrafted at the deep muscle layer of the tongue defect ( ); ∼10% engrafted PKH26 labeled GMSCs harbored overlapping green signals that represent the positive expression of MyoD, Myf5, or PAX7 ( ).

    Techniques: Expressing, Staining, Fluorescence, Microscopy, Transplantation Assay, Immunostaining

    Increased expression of MyoD and PAX7 in both wounded tongue areas with transplantation of GMSC/SIS-ECM and SIS-ECM in comparison with defect control. Expression of MyoD ( green ) (A) and PAX7 ( green ) (D) at local defect area 2 weeks after surgery in control, SIS-ECM only and GMSC/SIS-ECMs construct groups were determined by immunostaining and observed under a fluorescence microscope. The nuclei were counterstained with DAPI (original magnification ×10; ×20). The integrated immunofluorescence density of a ROI for both MyoD (B) and PAX7 (E) was quantified using Olympus cellSens™ imaging software. (C, F) The expression of MyoD and PAX7 proteins at the injured areas of the tongue was determined by Western blot analysis. ROI, region of interest.

    Journal: Tissue Engineering. Part A

    Article Title: A Gingiva-Derived Mesenchymal Stem Cell-Laden Porcine Small Intestinal Submucosa Extracellular Matrix Construct Promotes Myomucosal Regeneration of the Tongue

    doi: 10.1089/ten.tea.2016.0342

    Figure Lengend Snippet: Increased expression of MyoD and PAX7 in both wounded tongue areas with transplantation of GMSC/SIS-ECM and SIS-ECM in comparison with defect control. Expression of MyoD ( green ) (A) and PAX7 ( green ) (D) at local defect area 2 weeks after surgery in control, SIS-ECM only and GMSC/SIS-ECMs construct groups were determined by immunostaining and observed under a fluorescence microscope. The nuclei were counterstained with DAPI (original magnification ×10; ×20). The integrated immunofluorescence density of a ROI for both MyoD (B) and PAX7 (E) was quantified using Olympus cellSens™ imaging software. (C, F) The expression of MyoD and PAX7 proteins at the injured areas of the tongue was determined by Western blot analysis. ROI, region of interest.

    Article Snippet: At day 14 post-transplantation of the PKH26-labeled GMSC/SIS-ECM construct, frozen sections were harvested and immunostained with antibodies for MyoD, Myf5, and PAX7, three myogenic transcription factors., Using immunofluorescent studies, we showed that PKH26 labeled GMSCs transplanted with SIS-ECM scaffold engrafted at the deep muscle layer of the tongue defect ( ); ∼10% engrafted PKH26 labeled GMSCs harbored overlapping green signals that represent the positive expression of MyoD, Myf5, or PAX7 ( ).

    Techniques: Expressing, Transplantation Assay, Construct, Immunostaining, Fluorescence, Microscopy, Immunofluorescence, Imaging, Software, Western Blot

    Reconstitution of the Satellite Cell Niche by Donor MABs in IM Transplanted Muscles (A) Immunofluorescence staining images showing Pax7 staining for satellite cells and β-galactosidase staining for transplanted mdx (DYS nLacZ PB) MABs. Top panels show the co-localization of a Pax7 and β-galactosidase staining of positive nucleus under the basal lamina (MAB-derived satellite cell). Bottom panel shows a Pax7 positive nucleus that is not β-galactosidase positive under the basal lamina (resident satellite cell). Scale bar, 20 μm. (B) Fluorescence-based cell sorting of GFP-positive satellite cells ( mdx DYS GFP PB MAB-derived) and GFP-negative (resident) satellite cells. Boxes in all left panels highlight the proportion of Sca-1 − and Integrin-α7 + cells among the subpopulation of CD31 − , CD45 − , and CD11b − mononuclear cells, among which the GFP + cells are boxed on right-hand side panels, i.e., there are 3.17% of GFP + cells among the Sca-1 − , Integrin-α7 + , CD31 − , CD45 − , CD11b − mononuclear cells isolated from the mdx SCID mouse muscles after three transplantations. (C) Averages of the percentage of GFP + satellite cells derived from transplanted mdx (DYS GFP PB) MABs from the total population of satellite cells in mdx/ SCID three-transplant mice, isolated 6 weeks after last transplant, and mdx/ SCID single transplant 24 weeks after transplantation. Values are expressed as mean ± SD of values from different muscles used for satellite cell isolation (n = 4 for each condition). (D) qPCR analysis of transcript levels of the Pax7 and integrin-α7 satellite cell markers in the resident satellite cell population and donor MAB-derived satellite cells of transplanted muscles; n.d., not detectable.

    Journal: Molecular Therapy

    Article Title: Autologous Cell Therapy Approach for Duchenne Muscular Dystrophy using PiggyBac Transposons and Mesoangioblasts

    doi: 10.1016/j.ymthe.2018.01.021

    Figure Lengend Snippet: Reconstitution of the Satellite Cell Niche by Donor MABs in IM Transplanted Muscles (A) Immunofluorescence staining images showing Pax7 staining for satellite cells and β-galactosidase staining for transplanted mdx (DYS nLacZ PB) MABs. Top panels show the co-localization of a Pax7 and β-galactosidase staining of positive nucleus under the basal lamina (MAB-derived satellite cell). Bottom panel shows a Pax7 positive nucleus that is not β-galactosidase positive under the basal lamina (resident satellite cell). Scale bar, 20 μm. (B) Fluorescence-based cell sorting of GFP-positive satellite cells ( mdx DYS GFP PB MAB-derived) and GFP-negative (resident) satellite cells. Boxes in all left panels highlight the proportion of Sca-1 − and Integrin-α7 + cells among the subpopulation of CD31 − , CD45 − , and CD11b − mononuclear cells, among which the GFP + cells are boxed on right-hand side panels, i.e., there are 3.17% of GFP + cells among the Sca-1 − , Integrin-α7 + , CD31 − , CD45 − , CD11b − mononuclear cells isolated from the mdx SCID mouse muscles after three transplantations. (C) Averages of the percentage of GFP + satellite cells derived from transplanted mdx (DYS GFP PB) MABs from the total population of satellite cells in mdx/ SCID three-transplant mice, isolated 6 weeks after last transplant, and mdx/ SCID single transplant 24 weeks after transplantation. Values are expressed as mean ± SD of values from different muscles used for satellite cell isolation (n = 4 for each condition). (D) qPCR analysis of transcript levels of the Pax7 and integrin-α7 satellite cell markers in the resident satellite cell population and donor MAB-derived satellite cells of transplanted muscles; n.d., not detectable.

    Article Snippet: Samples were then immersed in sodium citrate buffer (10 mM [pH 6.0]) at 100°C for demasking the antigen epitopes and incubated overnight at 4°C with anti-mouse Pax7.

    Techniques: Immunofluorescence, Staining, Derivative Assay, Fluorescence, FACS, Isolation, Mouse Assay, Transplantation Assay, Cell Isolation, Real-time Polymerase Chain Reaction

    Aged SCs exhibit defective activation of ERK1/2, p38 MAPK and Akt and low expression levels of the transcription factors, MyoD and Pax7. a Young and aged SCs were cultivated in GM or DM and subjected to Western blotting using anti-phosphorylated p38 MAPK,

    Journal: Age

    Article Title: Human muscle satellite cells show age-related differential expression of S100B protein and RAGE

    doi: 10.1007/s11357-010-9197-x

    Figure Lengend Snippet: Aged SCs exhibit defective activation of ERK1/2, p38 MAPK and Akt and low expression levels of the transcription factors, MyoD and Pax7. a Young and aged SCs were cultivated in GM or DM and subjected to Western blotting using anti-phosphorylated p38 MAPK,

    Article Snippet: Then, the cells were incubated overnight at 4°C with a monoclonal anti-Pax7 (1:20, R & D Systems), monoclonal anti-MyoD (1:10, Santa Cruz Biotechnology), or monoclonal anti-myogenin (1:20, Santa Cruz Biotechnology) antibody in combination with the polyclonal anti-S100B (1:30, SWant) antibody.

    Techniques: Activation Assay, Expressing, Western Blot

    S100B, bFGF and HMGB1 are released from injured muscles. ( A ) CM from uninjured or BaCl2-injured Tibialis anterior muscles were processed for detection of S100B, bFGF and HMGB1 by Western blotting. S100B, bFGF and HMGB1 in CM were quantified by comparing the density of individual western blot bands in the CM with that of increasing amounts of each purified protein in parallel western blots. ( B ) After collection of CM, muscle tissue was homogenized and subjected to Western blotting for detection of RAGE and FGFR1 (20 µg protein loaded/lane). For experiments in A and B three animals/time point were used and the whole experiment was performed two times. Results in A and B are expressed as means ± SD. C, Freshly excised uninjured or BaCl 2 -injured Tibialis anterior muscles were processed for detection of Pax7 and RAGE by double-immunofluorescence. Nuclei were counterstained with DAPI. RAGE is not expressed in uninjured tissue. Arrow points to a quiescent satellite (Pax7 + ) cell (a,a′) and to a Pax7 + /RAGE + myoblast outside regenerating myofibers (b,b′). Arrowhead points to a Pax7 + /RAGE + myoblast within a regenerating myofiber (b,b′). Bars = 100 µm.

    Journal: PLoS ONE

    Article Title: S100B Engages RAGE or bFGF/FGFR1 in Myoblasts Depending on Its Own Concentration and Myoblast Density. Implications for Muscle Regeneration

    doi: 10.1371/journal.pone.0028700

    Figure Lengend Snippet: S100B, bFGF and HMGB1 are released from injured muscles. ( A ) CM from uninjured or BaCl2-injured Tibialis anterior muscles were processed for detection of S100B, bFGF and HMGB1 by Western blotting. S100B, bFGF and HMGB1 in CM were quantified by comparing the density of individual western blot bands in the CM with that of increasing amounts of each purified protein in parallel western blots. ( B ) After collection of CM, muscle tissue was homogenized and subjected to Western blotting for detection of RAGE and FGFR1 (20 µg protein loaded/lane). For experiments in A and B three animals/time point were used and the whole experiment was performed two times. Results in A and B are expressed as means ± SD. C, Freshly excised uninjured or BaCl 2 -injured Tibialis anterior muscles were processed for detection of Pax7 and RAGE by double-immunofluorescence. Nuclei were counterstained with DAPI. RAGE is not expressed in uninjured tissue. Arrow points to a quiescent satellite (Pax7 + ) cell (a,a′) and to a Pax7 + /RAGE + myoblast outside regenerating myofibers (b,b′). Arrowhead points to a Pax7 + /RAGE + myoblast within a regenerating myofiber (b,b′). Bars = 100 µm.

    Article Snippet: Sections were washed with TBS, pH 7.4, incubated for 1 h with Blocking Buffer (BB, 0.4% Triton-X-100, 10% donkey serum and 1% bovine serum albumin in phosphate-buffered saline) and then probed with the following primary antibodies in BB (1∶20): goat polyclonal anti-RAGE (Santa Cruz Biotechnology), mouse monoclonal anti-Pax7 (R & D Systems).

    Techniques: Western Blot, Purification, Immunofluorescence

    Characterization of iPS clone—TTF2. ( a ) Morphology of iPax7-iPS clone. ( b ) Western blot analyses for Pax7 in iPax7-iPS clones TTF2 and ICE7. ( c ) FACS analysis for SSEA-1 expression. ( d ) Immunofluorescent staining for Nanog. ( e ) Staining for alkaline

    Journal: Stem cell reviews

    Article Title: Functional Myogenic Engraftment from Mouse iPS Cells

    doi: 10.1007/s12015-011-9258-2

    Figure Lengend Snippet: Characterization of iPS clone—TTF2. ( a ) Morphology of iPax7-iPS clone. ( b ) Western blot analyses for Pax7 in iPax7-iPS clones TTF2 and ICE7. ( c ) FACS analysis for SSEA-1 expression. ( d ) Immunofluorescent staining for Nanog. ( e ) Staining for alkaline

    Article Snippet: Tet-inducible expression of Pax7 for both cell lines was assessed by western blot using a monoclonal anti-Pax7 antibody (R & D Systems).

    Techniques: Western Blot, Clone Assay, FACS, Expressing, Staining

    Scheme of the methods utilized to generate inducible Pax7 iPS cells. In one approach, iPS cells were derived from tail tip fibroblasts (TTF) obtained from an iPax7 mouse, which was generated following the blastocyst injection of iPax7 ES cells. In the

    Journal: Stem cell reviews

    Article Title: Functional Myogenic Engraftment from Mouse iPS Cells

    doi: 10.1007/s12015-011-9258-2

    Figure Lengend Snippet: Scheme of the methods utilized to generate inducible Pax7 iPS cells. In one approach, iPS cells were derived from tail tip fibroblasts (TTF) obtained from an iPax7 mouse, which was generated following the blastocyst injection of iPax7 ES cells. In the

    Article Snippet: Tet-inducible expression of Pax7 for both cell lines was assessed by western blot using a monoclonal anti-Pax7 antibody (R & D Systems).

    Techniques: Derivative Assay, Generated, Injection

    Engraftment ability of iPax7 iPS-derived myogenic progenitors into mdx mice. Pax7-induced (dox) cell monolayers resulting from PDGFαR + Flk-1 − sorted cells from day 5 iPax7 and iPax7-ICE iPS embryoid bodies were transplanted via intramuscular

    Journal: Stem cell reviews

    Article Title: Functional Myogenic Engraftment from Mouse iPS Cells

    doi: 10.1007/s12015-011-9258-2

    Figure Lengend Snippet: Engraftment ability of iPax7 iPS-derived myogenic progenitors into mdx mice. Pax7-induced (dox) cell monolayers resulting from PDGFαR + Flk-1 − sorted cells from day 5 iPax7 and iPax7-ICE iPS embryoid bodies were transplanted via intramuscular

    Article Snippet: Tet-inducible expression of Pax7 for both cell lines was assessed by western blot using a monoclonal anti-Pax7 antibody (R & D Systems).

    Techniques: Derivative Assay, Mouse Assay

    Pharmacological role of CB1 in primary human muscle cells. a Representative blot for CB1 and PAX7 protein expression in primary human satellite cells. The approximate molecular mass for each of these proteins (expressed in kDa) is shown on the right. b Bar graph showing the mRNA expression levels of MYOG and TNNT-1 in satellite cells exposed to DM for 5 days +/− rimonabant 1–3 µM. Each bar is the mean ± SEM of four separate determinations. c Morphological analysis of myotube formation in human primary satellite cells exposed to DM for 5 days in the presence of vehicle (DMSO, control) or rimonabant 1 µM. MyHC (red) and DAPI (blue). (Scale bar, 10 μm). The fusion index was calculated in both vehicle (DMSO)- and rimonabant-treated cells exposed to DM for 5 days. The asterisk denotes the P ≤ 0.05 vs. vehicle-treated cells. d Bar graphs showing the MYOG and TNNT-1 mRNA expression levels in primary human myoblasts isolated from each DMD donors (D1–D9) and induced to differentiate in presence of vehicle (DMSO) or rimonabant (1 µM). The quantification of transcripts was performed in quadruplicate by quantitative real-time PCR. The error bars correspond to the internal SEM. e The graph shows the differences in the expression levels of MYOG and TNNT-1 between vehicle- or rimonabant-treated myoblasts calculated by combining the DMD patient’s results together. Each bar is the mean ± SEM of the data from the nine patients, each of which was obtained from at least four separate determinations (see d ). * P ≤ 0.05 vs. vehicle group, determined by Student’s t test

    Journal: Nature Communications

    Article Title: Genetic and pharmacological regulation of the endocannabinoid CB1 receptor in Duchenne muscular dystrophy

    doi: 10.1038/s41467-018-06267-1

    Figure Lengend Snippet: Pharmacological role of CB1 in primary human muscle cells. a Representative blot for CB1 and PAX7 protein expression in primary human satellite cells. The approximate molecular mass for each of these proteins (expressed in kDa) is shown on the right. b Bar graph showing the mRNA expression levels of MYOG and TNNT-1 in satellite cells exposed to DM for 5 days +/− rimonabant 1–3 µM. Each bar is the mean ± SEM of four separate determinations. c Morphological analysis of myotube formation in human primary satellite cells exposed to DM for 5 days in the presence of vehicle (DMSO, control) or rimonabant 1 µM. MyHC (red) and DAPI (blue). (Scale bar, 10 μm). The fusion index was calculated in both vehicle (DMSO)- and rimonabant-treated cells exposed to DM for 5 days. The asterisk denotes the P ≤ 0.05 vs. vehicle-treated cells. d Bar graphs showing the MYOG and TNNT-1 mRNA expression levels in primary human myoblasts isolated from each DMD donors (D1–D9) and induced to differentiate in presence of vehicle (DMSO) or rimonabant (1 µM). The quantification of transcripts was performed in quadruplicate by quantitative real-time PCR. The error bars correspond to the internal SEM. e The graph shows the differences in the expression levels of MYOG and TNNT-1 between vehicle- or rimonabant-treated myoblasts calculated by combining the DMD patient’s results together. Each bar is the mean ± SEM of the data from the nine patients, each of which was obtained from at least four separate determinations (see d ). * P ≤ 0.05 vs. vehicle group, determined by Student’s t test

    Article Snippet: Filters were incubated overnight at 4 °C with the following primary antibodies: (a) mouse anti-CB1 (1:500; cat. no. Y080037, Applied Biological Materials Inc.); mouse anti-PAX7 (1:500; SantaCruz CA USA, cat. no. sc-81648); rabbit anti PKC-pan (phospho Thr497; 1:500; Thermo Fisher Scientific; cat. no. GTX52316); rabbit anti PKC-pan total (1:500; Thermo Fisher Scientific; cat. no. GTX52352).

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction

    CB1 and PAX7 gene expression in dystrophic skeletal muscles and cells. a , b Time course of CB1 and PAX7 mRNA expression levels in gastrocnemius ( a ) and quadriceps ( b ) muscle of control (dark yellow columns) and mdx (red columns) mice of 3 ( n = 8), 5 ( n = 8), and 8 ( n = 8) weeks of age. The quantification of transcripts for CB1 and PAX7 was performed by quantitative real-time PCR. c Heatmap representation of selected genes obtained from RNA-seq analysis in fibroadipogenic (FAP), satellite (SC), and macrophage (MP) cells isolated from 8-week-old control ( n = 4) and mdx ( n = 4) mice. Red, upregulated; green, downregulated. d Bar graph showing RPKM (Reads Per Kilobase of transcript per Million mapped reads) normalized values for the CB1 gene obtained from RNA-seq analysis in isolated FAP, SC, and MP cells. Each bar is the mean ± SEM of the independent determinations. * P ≤ 0.05 vs. control animals or cells, determined by Student’s t test

    Journal: Nature Communications

    Article Title: Genetic and pharmacological regulation of the endocannabinoid CB1 receptor in Duchenne muscular dystrophy

    doi: 10.1038/s41467-018-06267-1

    Figure Lengend Snippet: CB1 and PAX7 gene expression in dystrophic skeletal muscles and cells. a , b Time course of CB1 and PAX7 mRNA expression levels in gastrocnemius ( a ) and quadriceps ( b ) muscle of control (dark yellow columns) and mdx (red columns) mice of 3 ( n = 8), 5 ( n = 8), and 8 ( n = 8) weeks of age. The quantification of transcripts for CB1 and PAX7 was performed by quantitative real-time PCR. c Heatmap representation of selected genes obtained from RNA-seq analysis in fibroadipogenic (FAP), satellite (SC), and macrophage (MP) cells isolated from 8-week-old control ( n = 4) and mdx ( n = 4) mice. Red, upregulated; green, downregulated. d Bar graph showing RPKM (Reads Per Kilobase of transcript per Million mapped reads) normalized values for the CB1 gene obtained from RNA-seq analysis in isolated FAP, SC, and MP cells. Each bar is the mean ± SEM of the independent determinations. * P ≤ 0.05 vs. control animals or cells, determined by Student’s t test

    Article Snippet: Filters were incubated overnight at 4 °C with the following primary antibodies: (a) mouse anti-CB1 (1:500; cat. no. Y080037, Applied Biological Materials Inc.); mouse anti-PAX7 (1:500; SantaCruz CA USA, cat. no. sc-81648); rabbit anti PKC-pan (phospho Thr497; 1:500; Thermo Fisher Scientific; cat. no. GTX52316); rabbit anti PKC-pan total (1:500; Thermo Fisher Scientific; cat. no. GTX52352).

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Isolation

    CB1 and PAX7 expression and 2-AG levels in dystrophic muscles. a mRNA expression levels of CB1 and PAX7 in muscle samples of healthy children (HT, controls n = 3) or DMD donors of 3 ( n = 4) and 7 ( n = 4) years. b Measurement of the endogenous levels of 2-AG in muscle samples of the same healthy and DMD donors. Time course of the endogenous levels of 2-AG in the quadriceps ( c ) and gastrocnemius ( d ) muscle of control (dark yellow; n = 8) and mdx (red columns; n = 8) mice. The levels of 2-AG are expressed as pmol mg −1 of wet tissue weight. * P ≤ 0.05 vs. control group, determined by Student’s t test. e The bar graphs show RPKM (Reads Per Kilobase of transcript per Million mapped reads) normalized values for the Daglα and Magl genes obtained from RNA-seq analysis in satellite cells isolated from 8-week-old wt ( n = 4) and mdx mice ( n = 4)

    Journal: Nature Communications

    Article Title: Genetic and pharmacological regulation of the endocannabinoid CB1 receptor in Duchenne muscular dystrophy

    doi: 10.1038/s41467-018-06267-1

    Figure Lengend Snippet: CB1 and PAX7 expression and 2-AG levels in dystrophic muscles. a mRNA expression levels of CB1 and PAX7 in muscle samples of healthy children (HT, controls n = 3) or DMD donors of 3 ( n = 4) and 7 ( n = 4) years. b Measurement of the endogenous levels of 2-AG in muscle samples of the same healthy and DMD donors. Time course of the endogenous levels of 2-AG in the quadriceps ( c ) and gastrocnemius ( d ) muscle of control (dark yellow; n = 8) and mdx (red columns; n = 8) mice. The levels of 2-AG are expressed as pmol mg −1 of wet tissue weight. * P ≤ 0.05 vs. control group, determined by Student’s t test. e The bar graphs show RPKM (Reads Per Kilobase of transcript per Million mapped reads) normalized values for the Daglα and Magl genes obtained from RNA-seq analysis in satellite cells isolated from 8-week-old wt ( n = 4) and mdx mice ( n = 4)

    Article Snippet: Filters were incubated overnight at 4 °C with the following primary antibodies: (a) mouse anti-CB1 (1:500; cat. no. Y080037, Applied Biological Materials Inc.); mouse anti-PAX7 (1:500; SantaCruz CA USA, cat. no. sc-81648); rabbit anti PKC-pan (phospho Thr497; 1:500; Thermo Fisher Scientific; cat. no. GTX52316); rabbit anti PKC-pan total (1:500; Thermo Fisher Scientific; cat. no. GTX52352).

    Techniques: Expressing, Mouse Assay, RNA Sequencing Assay, Isolation

    Luciferase assay in PAX7-transfected HEK293 cells. Relative luciferase activity following transfection of human constructs into HEK293 cells. Cells were co-transfected with the pGL3 promoter vector carrying the construct of interest (regions 2, 3, and 4) cloned upstream of the luciferase gene together with an equimolar amount of plasmid encoding for human PAX7. For the control condition, PAX7 plasmid was replaced with pCDNA3.1. Cells were co-transfected with a β-Gal encoding vector to normalize transfection efficiency and the signal intensity of luciferase. The inset on the right shows a representative chemiluminescent signal emitted from the reaction of luciferase from the different experimental conditions. Data are expressed as the mean ± SEM of four independent determinations. * P ≤ 0.05 vs. control group, determined by Student’s t test

    Journal: Nature Communications

    Article Title: Genetic and pharmacological regulation of the endocannabinoid CB1 receptor in Duchenne muscular dystrophy

    doi: 10.1038/s41467-018-06267-1

    Figure Lengend Snippet: Luciferase assay in PAX7-transfected HEK293 cells. Relative luciferase activity following transfection of human constructs into HEK293 cells. Cells were co-transfected with the pGL3 promoter vector carrying the construct of interest (regions 2, 3, and 4) cloned upstream of the luciferase gene together with an equimolar amount of plasmid encoding for human PAX7. For the control condition, PAX7 plasmid was replaced with pCDNA3.1. Cells were co-transfected with a β-Gal encoding vector to normalize transfection efficiency and the signal intensity of luciferase. The inset on the right shows a representative chemiluminescent signal emitted from the reaction of luciferase from the different experimental conditions. Data are expressed as the mean ± SEM of four independent determinations. * P ≤ 0.05 vs. control group, determined by Student’s t test

    Article Snippet: Filters were incubated overnight at 4 °C with the following primary antibodies: (a) mouse anti-CB1 (1:500; cat. no. Y080037, Applied Biological Materials Inc.); mouse anti-PAX7 (1:500; SantaCruz CA USA, cat. no. sc-81648); rabbit anti PKC-pan (phospho Thr497; 1:500; Thermo Fisher Scientific; cat. no. GTX52316); rabbit anti PKC-pan total (1:500; Thermo Fisher Scientific; cat. no. GTX52352).

    Techniques: Luciferase, Transfection, Activity Assay, Construct, Plasmid Preparation, Clone Assay

    Bioinformatics and ChIP analysis. Schematic representation of the human ( a ) or murine ( b ) CB1 gene. The transcriptional start site (TSS) for each gene isoform is indicated. The identified PAX7 sites are shown as small black rectangles below the schematic. Regions identified as 1–4, indicated by red arrows, correspond to PAX7-containing gene regions of high sequence homology in human and murine CB1 gene. c PAX7 occupancy of identified sites in the CB1 gene evaluated by chromatin immunoprecipitation (ChIP) analysis. Left: average data of the relative amount of the PAX7-immunoprecipitated DNA in the murine quadriceps muscle isolated from either wild-type or mdx mice; Right: average data of the relative amount of the PAX7-immunoprecipitated DNA in PAX7-silenced satellite cells. Data are from six separate experiments and normalized relative to the input DNA. The inset shows a representative agarose gel electrophoresis of the qPCR products obtained from PAX7-immunoprecipitated DNA for each experimental condition. d Left: representative agarose gel electrophoresis of the qPCR products obtained from PAX7-immunoprecipitated DNA for each experimental condition. Right: average data of the relative amount of the PAX7-immunoprecipitated DNA in HEK293 cells transfected with control scramble ( n = 4) human PAX7 construct ( n = 4). * P ≤ 0.05 vs. control group, determined by Student’s t test

    Journal: Nature Communications

    Article Title: Genetic and pharmacological regulation of the endocannabinoid CB1 receptor in Duchenne muscular dystrophy

    doi: 10.1038/s41467-018-06267-1

    Figure Lengend Snippet: Bioinformatics and ChIP analysis. Schematic representation of the human ( a ) or murine ( b ) CB1 gene. The transcriptional start site (TSS) for each gene isoform is indicated. The identified PAX7 sites are shown as small black rectangles below the schematic. Regions identified as 1–4, indicated by red arrows, correspond to PAX7-containing gene regions of high sequence homology in human and murine CB1 gene. c PAX7 occupancy of identified sites in the CB1 gene evaluated by chromatin immunoprecipitation (ChIP) analysis. Left: average data of the relative amount of the PAX7-immunoprecipitated DNA in the murine quadriceps muscle isolated from either wild-type or mdx mice; Right: average data of the relative amount of the PAX7-immunoprecipitated DNA in PAX7-silenced satellite cells. Data are from six separate experiments and normalized relative to the input DNA. The inset shows a representative agarose gel electrophoresis of the qPCR products obtained from PAX7-immunoprecipitated DNA for each experimental condition. d Left: representative agarose gel electrophoresis of the qPCR products obtained from PAX7-immunoprecipitated DNA for each experimental condition. Right: average data of the relative amount of the PAX7-immunoprecipitated DNA in HEK293 cells transfected with control scramble ( n = 4) human PAX7 construct ( n = 4). * P ≤ 0.05 vs. control group, determined by Student’s t test

    Article Snippet: Filters were incubated overnight at 4 °C with the following primary antibodies: (a) mouse anti-CB1 (1:500; cat. no. Y080037, Applied Biological Materials Inc.); mouse anti-PAX7 (1:500; SantaCruz CA USA, cat. no. sc-81648); rabbit anti PKC-pan (phospho Thr497; 1:500; Thermo Fisher Scientific; cat. no. GTX52316); rabbit anti PKC-pan total (1:500; Thermo Fisher Scientific; cat. no. GTX52352).

    Techniques: Chromatin Immunoprecipitation, Sequencing, Immunoprecipitation, Isolation, Mouse Assay, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Transfection, Construct

    Loss of function of PGE2 signaling in MuSCs impairs muscle regeneration and strength. ( A–H ) TAs of Pax7-specific EP4 conditional knockout mice ( Pax7 CreERT2 ;EP4 f/f , EP4 cKO) treated with tamoxifen (TAM) were assayed at 7 ( C and E ), 14 ( G and H

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Prostaglandin E2 is essential for efficacious skeletal muscle stem-cell function, augmenting regeneration and strength

    doi: 10.1073/pnas.1705420114

    Figure Lengend Snippet: Loss of function of PGE2 signaling in MuSCs impairs muscle regeneration and strength. ( A–H ) TAs of Pax7-specific EP4 conditional knockout mice ( Pax7 CreERT2 ;EP4 f/f , EP4 cKO) treated with tamoxifen (TAM) were assayed at 7 ( C and E ), 14 ( G and H

    Article Snippet: For mouse injury assays, we incubated transverse sections with Rabbit polyclonal anti-PGE2 (abcam, catalog no. ab2318, 1:100), rat polyclonal anti-Laminin (Clone A5) (EMD Millipore, catalog no. 05–206, 1:200), mouse monoclonal anti-Pax7 (Santa Cruz, catalog no. sc-81648, 1:50), AlexaFluor 647-conjugated wheat germ agglutinin (WGA) antibody ( , Thermo Fisher Scientific), rabbit polyclonal anti-GFP ( , Thermo Fisher Scientific, 1:500), and mouse monoclonal anti-eMyHC (DSHB, catalog no. F1.652, 1:10) primary antibodies and then with AlexaFluor secondary antibodies (Jackson ImmunoResearch Laboratories, 1:500).

    Techniques: Knock-Out, Mouse Assay

    dKO Satellite Cells Express Markers of Myogenic Commitment after Muscle Injury (A–F) PAX7 immunofluorescence of transverse cryosections of the TA muscle 31 dpi. PAX7+ cells (examples at yellow arrowheads) carrying a wild-type allele of MyoD (A and D) or Myf5 (B and E) were associated with regenerated fibers, identified by their central nucleation (examples at arrows). PAX7+ dKO cells were numerous at 31 dpi (C and F). Apparent PAX7 levels were reduced in dKO satellite cells. Exposure times were identical between genotypes. Scale bars represent 10 μm. (G–I) qRT-PCR analysis of myogenic gene expression of FACS-isolated satellite cells. ΔCt values were calculated using the average Ct values of the internal controls, Gapdh and EiF1a , which were highly consistent between genotypes within a given stage. Average Ct values were as follows: uninjured, 23.75; 3 dpi, 20.92; 12 dpi, 24.54. Each data point represents the average value of three biological samples and three technical replicates for each sample. Error bars represent standard deviations. p

    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 Satellite Cells Express Markers of Myogenic Commitment after Muscle Injury (A–F) PAX7 immunofluorescence of transverse cryosections of the TA muscle 31 dpi. PAX7+ cells (examples at yellow arrowheads) carrying a wild-type allele of MyoD (A and D) or Myf5 (B and E) were associated with regenerated fibers, identified by their central nucleation (examples at arrows). PAX7+ dKO cells were numerous at 31 dpi (C and F). Apparent PAX7 levels were reduced in dKO satellite cells. Exposure times were identical between genotypes. Scale bars represent 10 μm. (G–I) qRT-PCR analysis of myogenic gene expression of FACS-isolated satellite cells. ΔCt values were calculated using the average Ct values of the internal controls, Gapdh and EiF1a , which were highly consistent between genotypes within a given stage. Average Ct values were as follows: uninjured, 23.75; 3 dpi, 20.92; 12 dpi, 24.54. Each data point represents the average value of three biological samples and three technical replicates for each sample. Error bars represent standard deviations. p

    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: Immunofluorescence, Quantitative RT-PCR, Expressing, FACS, Isolation

    dKO Satellite Cells Can Adopt an Adipogenic or Fibroblast-like Fate Following Muscle Injury (A–H) Immunofluorescence localization of PAX7 and PLIN1 on transverse cryosections of TA muscles collected 6 dpi. Some dKO satellite cells gave rise to GFP+ PLIN1+ adipocytes (E, F, and H); examples at white arrowheads, whereas MyoD-SA satellite cells did not adopt an adipogenic fate (A, B, and D). Representative PAX7+ cells are shown with yellow arrowheads. Arrows in (E, F, and H), represent examples of GFP–, PLIN1+ adipocytes. (I–L) Picrosirius red staining of TA cross-sections at 11 dpi. Scale bars represent 25 μm (A and E) and 50 μm (I–L). (M–O) qRT-PCR analysis of fibrogenic gene expression of freshly isolated satellite cells. Internal controls and methods used to calculate ΔCt values are described in the legend to Figure 4 . Each data point represents the average value of three biological samples and three technical replicates for each sample. Error bars represent standard deviations. p

    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 Satellite Cells Can Adopt an Adipogenic or Fibroblast-like Fate Following Muscle Injury (A–H) Immunofluorescence localization of PAX7 and PLIN1 on transverse cryosections of TA muscles collected 6 dpi. Some dKO satellite cells gave rise to GFP+ PLIN1+ adipocytes (E, F, and H); examples at white arrowheads, whereas MyoD-SA satellite cells did not adopt an adipogenic fate (A, B, and D). Representative PAX7+ cells are shown with yellow arrowheads. Arrows in (E, F, and H), represent examples of GFP–, PLIN1+ adipocytes. (I–L) Picrosirius red staining of TA cross-sections at 11 dpi. Scale bars represent 25 μm (A and E) and 50 μm (I–L). (M–O) qRT-PCR analysis of fibrogenic gene expression of freshly isolated satellite cells. Internal controls and methods used to calculate ΔCt values are described in the legend to Figure 4 . Each data point represents the average value of three biological samples and three technical replicates for each sample. Error bars represent standard deviations. p

    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: Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Isolation

    Cultured dKO Cells Show Evidence of Altered Myogenic Programming (A–X) Cultured dKO satellite cells adopt a fibroblast-like appearance but continue to express PAX7 and desmin (DES). (A–D) Phase images of dKO and dHet satellite cells after 3 or 7 days in culture. (E–L) Immunofluorescence analysis of PAX7 expression in dHet (E, F, I, and J) and dKO (G, H, K, and L) satellite cells after culturing for 3 (E–H) or 21 (I–L) days in GM. Occasional PAX7– cells were observed at day 21 (arrows) (K and L). (M–P) dKO cells maintained PAX7 expression after 21 days in DM. In dHet cells (M and N), PAX7 was downregulated in myotubes (arrows), but maintained in mononuclear cells (examples at arrowheads). Arrows in (O) and (P), example of an occasional PAX7– dKO cell. (Q–X) Comparison of DES expression in dHet (Q, R, U, and V) and dKO (S, T, W, and X) satellite cells 3 or 14 days in GM. dKO cells lacking detectable DES were common by day 14 (X), arrows. (Y–B′) dKO satellite cells show a propensity for adipogenic differentiation. dKO cells were cultured in GM for 7 days and switched to DM for 13 (Y and Z) or 21 (A’ and B’) days. Approximately 10% of cells accumulated lipid droplets and were PLIN1+; examples at arrows in (Y and Z). Inset in (Z), phase contrast image of boxed lipid-filled cell in (Y) and (Z). PAX7 is downregulated in adipogenic cells; arrows in (A’) and (B’). Exposure times (ms) are shown in some panels to allow comparisons of apparent expression levels between genotypes. Scale bars represent 50 μm.

    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: Cultured dKO Cells Show Evidence of Altered Myogenic Programming (A–X) Cultured dKO satellite cells adopt a fibroblast-like appearance but continue to express PAX7 and desmin (DES). (A–D) Phase images of dKO and dHet satellite cells after 3 or 7 days in culture. (E–L) Immunofluorescence analysis of PAX7 expression in dHet (E, F, I, and J) and dKO (G, H, K, and L) satellite cells after culturing for 3 (E–H) or 21 (I–L) days in GM. Occasional PAX7– cells were observed at day 21 (arrows) (K and L). (M–P) dKO cells maintained PAX7 expression after 21 days in DM. In dHet cells (M and N), PAX7 was downregulated in myotubes (arrows), but maintained in mononuclear cells (examples at arrowheads). Arrows in (O) and (P), example of an occasional PAX7– dKO cell. (Q–X) Comparison of DES expression in dHet (Q, R, U, and V) and dKO (S, T, W, and X) satellite cells 3 or 14 days in GM. dKO cells lacking detectable DES were common by day 14 (X), arrows. (Y–B′) dKO satellite cells show a propensity for adipogenic differentiation. dKO cells were cultured in GM for 7 days and switched to DM for 13 (Y and Z) or 21 (A’ and B’) days. Approximately 10% of cells accumulated lipid droplets and were PLIN1+; examples at arrows in (Y and Z). Inset in (Z), phase contrast image of boxed lipid-filled cell in (Y) and (Z). PAX7 is downregulated in adipogenic cells; arrows in (A’) and (B’). Exposure times (ms) are shown in some panels to allow comparisons of apparent expression levels between genotypes. Scale bars represent 50 μm.

    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: Cell Culture, Immunofluorescence, Expressing, Mass Spectrometry

    dKO Satellite Cells Are Maintained with Aging (A) Schematic representation of tamoxifen/cardiotoxin administration schedule. (B–D) Merged images of gastrocnemius cryosections stained for PAX7 (green; arrowheads), laminin (red), and DAPI (blue). (E–H) Cross-sections of injured Myf5-SA (E and G) or dKO (F and H) TA muscles 11 dpi. Sections were stained with Picrosirius red to detect collagen accumulations (E and F) or represent merged GFP/DAPI images (G and H). The exposure time to capture the GFP signal was three times longer in (H) than in (G). Rare nascent fibers are labeled with arrowheads in (F) and (H). (I–K) Cryosections of uninjured TA muscles stained with Picrosirius red. (L–N) Low-magnification micrographs of uninjured gastrocnemius muscles imaged for GFP and DAPI. Examples of lineage-labeled GFP+ myofibers are shown (L and M, arrowheads). Scale bars represent 25 µm (B–F), 50 μm (G–K), and 200 µm (L–N). See also Figure S6 .

    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 Satellite Cells Are Maintained with Aging (A) Schematic representation of tamoxifen/cardiotoxin administration schedule. (B–D) Merged images of gastrocnemius cryosections stained for PAX7 (green; arrowheads), laminin (red), and DAPI (blue). (E–H) Cross-sections of injured Myf5-SA (E and G) or dKO (F and H) TA muscles 11 dpi. Sections were stained with Picrosirius red to detect collagen accumulations (E and F) or represent merged GFP/DAPI images (G and H). The exposure time to capture the GFP signal was three times longer in (H) than in (G). Rare nascent fibers are labeled with arrowheads in (F) and (H). (I–K) Cryosections of uninjured TA muscles stained with Picrosirius red. (L–N) Low-magnification micrographs of uninjured gastrocnemius muscles imaged for GFP and DAPI. Examples of lineage-labeled GFP+ myofibers are shown (L and M, arrowheads). Scale bars represent 25 µm (B–F), 50 μm (G–K), and 200 µm (L–N). See also Figure S6 .

    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: Staining, Labeling

    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