Journal: Journal of Molecular Endocrinology

Article Title: C2C12 myoblastoma cell differentiation and proliferation is stimulated by androgens and associated with a modulation of myostatin and Pax7 expression

doi: 10.1677/jme-07-0175

Figure Lengend Snippet: Figure 6 Analysis of Pax7 expression during C2C12 cell differentiation. (A) Time-dependent analysis of Pax7 protein expression. After 2 (d2), 4 (d4), 6 (d6), and 8 (d8) days cells were fixed and stained with a Pax7-specific antibody and DAPI. The analysis of expression was performed by confocal microscopy (magnification 200!). (B) Calculation of the percentage of Pax7-positive nuclei. Indicated are the days of culture in FCS medium. *Means statistically different against day 2 (P!0.05).

Article Snippet: The following antibodies were used: myostatin MF408 (R&D Systems, Wiesbaden, Germany), anti-goat T-7028TRITC conjugate (Sigma– Aldrich), paired box gene 7 (Pax7) antibody MAB 1675 (R&D Systems), and biotinylated goat anti-rabbit immunoglobulins E 0432/Dako, Hamburg, Germany).

Techniques: Expressing, Cell Differentiation, Staining, Confocal Microscopy

Journal: Journal of Molecular Endocrinology

Article Title: C2C12 myoblastoma cell differentiation and proliferation is stimulated by androgens and associated with a modulation of myostatin and Pax7 expression

doi: 10.1677/jme-07-0175

Figure Lengend Snippet: Figure 7 The effect of dihydrotestosterone (DHT) on Pax7 expression during C2C12 cell differentiation. (A) Time-dependent analysis of Pax7 protein expression. After 2 (d2), 4 (d4), 6 (d6), and 8 (d8) days, the cells were fixed and stained with a Pax7-specific antibody and DAPI (not shown). (B) Calculation of the percentage of Pax7-positive nuclei.

Article Snippet: The following antibodies were used: myostatin MF408 (R&D Systems, Wiesbaden, Germany), anti-goat T-7028TRITC conjugate (Sigma– Aldrich), paired box gene 7 (Pax7) antibody MAB 1675 (R&D Systems), and biotinylated goat anti-rabbit immunoglobulins E 0432/Dako, Hamburg, Germany).

Techniques: Expressing, Cell Differentiation, Staining

Journal: The FASEB Journal

Article Title: Correction of GSK3ß at young age prevents muscle pathology in mice with myotonic dystrophy type 1

doi: 10.1096/fj.201700700r

Figure Lengend Snippet: Figure 5. A) The percentage of genes for the functional pathways altered in skeletal muscle and in the brain of hom Celf1 KO mice. B) Confirmation of alterations of selected mRNAs identified by microarray analysis in skeletal muscle of Celf1 KO mice. RNA expression analysis of LEF1, DCX, RBM45, and Col4A in the hind-limb muscle of 1–4-d-old WT and Celf1 KO mice. LEF1, DCX, and RBM45 were analyzed in hom Celf1 KO muscle. Col4A mRNA was examined in het Celf1 KO muscle. C) Western blot analysis of LEF1, RBM45, Col 4A, and DCX in the gastroc from WT and Celf1+/2 littermates at 4 d old. b-actin was used as the control for protein loading. D) Western blot analysis of skeletal muscle from WT and Celf1+/2 littermates at 5 d and 12 mo old with antibodies to Pax7 and b-actin as control.

Article Snippet: Antibodies to actin (I-19), a-actinin (H-300), b-actin (C-4), calreticulin (H-170), cyclin D3 (C-16), collagen 4A (E-14), CUGBP1 (3B1), desmin (H-76), Pax7 (EE-8), phosphorylated (p-)S51-eIF2a (sc-101670), and troponin T-FS (H-8) (all from Santa Cruz Biotechnology, Dallas, TX, USA); GSK3b (3891) and DCX (4604) (Cell Signaling Technology, Danvers, MA, USA); LEF1 (Thermo Fisher Scientific, Waltham, MA, USA); and myosin skeletal fast (MY-32; MilliporeSigma, Billerica, MA, USA) were used.

Techniques: Functional Assay, Microarray, RNA Expression, Western Blot, Control

Journal: Life Science Alliance

Article Title: Metabolic reprogramming of fibro/adipogenic progenitors facilitates muscle regeneration

doi: 10.26508/lsa.202000660

Figure Lengend Snippet: List of key materials, reagents, animal strains, and software.

Article Snippet: Pax7 antibody (SPM613) (FITC) , Novus Biologicals , Cat. no. NBP2-47923F.

Techniques: Software, Recombinant, Western Blot, Membrane, Adhesive, Electron Microscopy, Modification, Cell Culture, Red Blood Cell Lysis, Protease Inhibitor, Imaging, Cell Viability Assay, Enzyme-linked Immunosorbent Assay, In Situ

Journal: Journal of Orthopaedic Translation

Article Title: Neuromuscular electrical stimulation alleviates stroke-related sarcopenia by promoting satellite cells myogenic differentiation via AMPK-ULK1-Autophagy axis

doi: 10.1016/j.jot.2025.03.021

Figure Lengend Snippet: NMES accelerated muscle regeneration and functional recovery in rats with stroke-related sarcopenia. (A) Catwalk Footprint Gait Analysis of rat hind limbs in the control and NMES groups eight weeks after surgery. The dotted line magnified the footprint and displayed the peak footprint intensity; scale bar: 1 cm. (B) Quantitative analysis of the mean intensity of the right/left hind foot and (C) left hind foot print area (n = 4 in the control group and n = 4 in the NMES group; ∗ P < 0.05, ∗∗∗∗ P < 0.0001 by two-tailed unpaired Student's t test). (D) Amplitude of CMAP and quantitative peak amplitude of CMAP (n = 5 in each group; ∗∗∗∗ P < 0.0001 by two-tailed unpaired Student's t test). (E) Target muscle general images and quantitative analysis showing muscle wet weight (n = 5 in each group; ∗ P < 0.05 by two-tailed unpaired Student's t test). (F) Immunofluorescence of Pax7 and Laminin in muscle with stroke-related sarcopenia from the control and NMES groups; scale bar: 100 μm. (G) Masson's trichrome staining of muscle eight weeks after surgery. Dotted squares indicated magnification zones; scale bar: 500 μm (for magnification, scale bar: 100 μm). (H) ImageJ software evaluated the number and average diameter (Ferret diameter) of muscle fibers in a random field of view from F-panel (n = 3 in each group; ∗ P < 0.05 and ns by two-tailed unpaired Student's t test). (I) Quantitative analysis of the myofiber, intramuscular space and collagen fiber in the Masson's trichrome staining from G-panel (n = 5 in each group; ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 by two-tailed unpaired Student's t test).

Article Snippet: Adherent cells were harvested at passage three for flow cytometry, where they were stained with fluorescence-labeled antibodies against Pax7 (NBP2-34706 PE, Novus, USA) and desmin (Y&M, BYK-1026R-FITC) ( ).

Techniques: Functional Assay, Control, Two Tailed Test, Immunofluorescence, Staining, Software

Journal: Journal of Orthopaedic Translation

Article Title: Neuromuscular electrical stimulation alleviates stroke-related sarcopenia by promoting satellite cells myogenic differentiation via AMPK-ULK1-Autophagy axis

doi: 10.1016/j.jot.2025.03.021

Figure Lengend Snippet: ES promoted myogenic differentiation of rat MuSCs. (A) Illustration of ES treatment in vitro . (B) Relative expression levels of muscle differentiation-related genes (Myf6, Myod1, Myom, Pax7, Fbxo32, Trim63) in MuSCs under different voltages for 30min measured by qRT-PCR (n = 3 in each group; relative to Gapdh; ∗ P < 0.05, ∗∗∗ P < 0.001 ∗∗∗∗ P < 0.0001 by one-way ANOVA, followed Bonferroni multiple comparisons post-hoc test). (C) Immunofluorescence staining for Myod1 (green), Pax7 (red) and DAPI (blue) in MuSCs under control and different voltages for 30min; scale bar: 200 μm. (magnification zones, scale bar: 50 μm) (D) Quantitative analysis of immunofluorescence intensity for Myod1 and Pax7 at various voltages (three random filed in each group; ∗∗∗∗ P < 0.0001 by one-way ANOVA and Bonferroni multiple comparisons post-hoc test). (E) MuSCs after ES treatment incubated in differentiation medium for three days, before immunostaining for myosin heavy chain (MyHC, MYH6) (green) and counterstained with DAPI (blue); scale bar: 200 μm. (F) Quantitative analysis of immunofluorescence intensity for MyHC of the E-panel (three random filed in each group; ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 by two-way ANOVA, followed by Tukey's multiple comparisons test)

Article Snippet: Adherent cells were harvested at passage three for flow cytometry, where they were stained with fluorescence-labeled antibodies against Pax7 (NBP2-34706 PE, Novus, USA) and desmin (Y&M, BYK-1026R-FITC) ( ).

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Control, Incubation, Immunostaining

Journal: Journal of Orthopaedic Translation

Article Title: Neuromuscular electrical stimulation alleviates stroke-related sarcopenia by promoting satellite cells myogenic differentiation via AMPK-ULK1-Autophagy axis

doi: 10.1016/j.jot.2025.03.021

Figure Lengend Snippet: AMPK mediated the myogenic role of ES. (A) KEGG enrichment analysis of DEGs (KEGG disease pathway filtered). (B) Immunofluorescence staining for p-Thr172-PRKAA, Pax7 and DAPI in muscle with stroke-related sarcopenia from control group and NMES-treated group; scale bar: 50 μm. (C) Western Blot analysis of PRKAA activation in MuSCs under control and different voltages for 30min. (D) Western Blot analysis of PRKAA activation in MuSCs pretreated with PRKAA inhibitor BML-275 (0.2 μM) or agonist AICAR (15 μM) for 24h in control group and ES treatment (5V treated for 30min) group. (E) Immunofluorescence staining of MuSCs after myotube differentiation for three days with BML-275 or AICAR in control group and ES treatment group. MyHC (green) and DAPI (blue) for myotube; scale bar: 200 μm (magnification zones, scale bar: 50 μm). (F) Quantitative analysis of immunofluorescence intensity for MyHC of the E-panel (three random filed in each group; ∗∗∗∗ P < 0.0001 by two-way ANOVA, followed by Tukey's multiple comparisons test). (G) Immunofluorescence staining of MuSCs after myotube differentiation for three days infected with scramble or shPrkaa1 lentivirus in control group and ES treatment group; scale bar: 200 μm (magnification zones, scale bar: 50 μm). (H) Quantitative analysis of immunofluorescence intensity for MyHC of the G-panel (three random filed in each group; ∗∗∗∗ P < 0.0001 by two-way ANOVA, followed by Tukey's multiple comparisons test).

Article Snippet: Adherent cells were harvested at passage three for flow cytometry, where they were stained with fluorescence-labeled antibodies against Pax7 (NBP2-34706 PE, Novus, USA) and desmin (Y&M, BYK-1026R-FITC) ( ).

Techniques: Immunofluorescence, Staining, Control, Western Blot, Activation Assay, Infection

Journal: Journal of Orthopaedic Translation

Article Title: Neuromuscular electrical stimulation alleviates stroke-related sarcopenia by promoting satellite cells myogenic differentiation via AMPK-ULK1-Autophagy axis

doi: 10.1016/j.jot.2025.03.021

Figure Lengend Snippet: AMPK activation caused autophagy during ES-induced myogenic differentiation. (A) Transmission electron microscopy of MuSCs in control group and ES treatment group. Arrows indicated autophagosomes; scale bar: 2 μm. (B) Western Blot analysis of autophagic activation in MuSCs under control and different voltages for 30min. (C) Autophagic flux detection for MuSCs transfected with HBAD-mcherry-EGFP-LC3 in control group and ES treatment group; scale bar: 50 μm. (D) Quantitative analysis of the autophagic flux in B-panel (n = 5 in each group; ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 by two-tailed unpaired Student's t test). (E) Immunofluorescence staining for LC3B, Pax7 and DAPI in muscle with stroke-related sarcopenia from control and NMES-treated rats; scale bar: 100 μm. (F) Western Blot analysis of autophagic activation in the MuSCs treated with autophagy inhibitors chloroquine (CQ, 40 μM) or bafilomycin A1 (BafA1, 1.5 μM) under control and ES conditions. (G) Immunofluorescence staining of MuSCs after myotube differentiation for three days with CQ or BafA1 under control and ES conditions; MyHC (green) and DAPI (blue) for myotube; scale bar: 200 μm (magnification zones, scale bar: 50 μm). (H) Quantitative analysis of immunofluorescence intensity for MyHC of the G-panel (three random filed in each group; ∗∗∗∗ P < 0.0001 by two-way ANOVA, followed by Tukey's multiple comparisons test). (I) Immunofluorescence staining of MuSCs after myotube differentiation for three days infected with scramble or shAtg7 lentivirus in control group and ES treatment group; MyHC (green) and DAPI (blue) for myotube; scale bar: 200 μm (magnification zones, scale bar: 50 μm). (J) Quantitative analysis of immunofluorescence intensity for MyHC of the E-panel (three random filed in each group; ∗∗∗∗ P < 0.0001 by two-way ANOVA, followed by Tukey's multiple comparisons test).

Article Snippet: Adherent cells were harvested at passage three for flow cytometry, where they were stained with fluorescence-labeled antibodies against Pax7 (NBP2-34706 PE, Novus, USA) and desmin (Y&M, BYK-1026R-FITC) ( ).

Techniques: Activation Assay, Transmission Assay, Electron Microscopy, Control, Western Blot, Transfection, Two Tailed Test, Immunofluorescence, Staining, Infection

Journal: Journal of Orthopaedic Translation

Article Title: Neuromuscular electrical stimulation alleviates stroke-related sarcopenia by promoting satellite cells myogenic differentiation via AMPK-ULK1-Autophagy axis

doi: 10.1016/j.jot.2025.03.021

Figure Lengend Snippet: AMPK phosphorylation of ULK1 was required for ES-induced autophagy and myogenic differentiation. (A) Western Blot analysis of ULK1 activation in MuSCs under control and different voltages for 30min. (B) Immunofluorescence staining for p-Ser317-ULK1, Pax7 and DAPI in muscle with stroke-related sarcopenia from control and NMES-treated rats; scale bar: 50 μm. (C) MuSCs were treated under control and ES conditions (5V treated for 30min). Immunoprecipitation (IP) was performed with PRKAA antibody or pre-immune IgG. Immunoblotting (IB) was carried out to detect PRKAA and ULK1. (D) Western Blot analysis of AMPK-ULK1/autophagic activation in MuSCs treated with ULK1 inhibitor SBI-0206965 (SBI, 1 μM) or agonist BL-918 (15 μM) in control group and ES treatment group. (E) Immunofluorescence staining of MuSCs after myotube differentiation for three days with SBI or BL in control group and ES treatment group; MyHC (green) and DAPI (blue) for myotube; scale bar: 200 μm (magnification zones, scale bar: 50 μm). Quantitative analysis of immunofluorescence intensity for MyHC of the E-panel (three random filed in each group; ∗ P < 0.05, ∗∗∗∗ P < 0.0001 by two-way ANOVA, followed by Tukey's multiple comparisons test). (F) Western Blot analysis of AMPK-ULK1/autophagic activation in rats using lentivirus vector-mediated shRNA (LV-Prkaa1shRNA, LV-Atg7shRNA and LV-Ulk1shRNA) in control group and NMES treatment group. (G) Immunofluorescence staining of MuSCs after myotube differentiation for three days infected with scramble or shUlk1 lentivirus in control group and ES treatment group; MyHC (green) and DAPI (blue) for myotube; scale bar: 200 μm (magnification zones, scale bar: 50 μm). Quantitative analysis of immunofluorescence intensity for MyHC of the G-panel (three random filed in each group; ∗∗∗∗ P < 0.0001 by two-way ANOVA, followed by Tukey's multiple comparisons test). (H) Western Blot analysis of AMPK-ULK1-autophagy activation in MuSCs with an PRKAA agonist AICAR (15 μM) or/and ULK1 inhibitor SBI (1 μM) in control group and ES treatment group. (I) Immunofluorescence staining of MuSCs after myotube differentiation for three days with AICAR or/and SBI under control and ES conditions; MyHC (green) and DAPI (blue) for myotube; scale bar: 200 μm (magnification zones, scale bar: 50 μm). Quantitative analysis of immunofluorescence intensity for MyHC of the E-panel (three random filed in each group; ∗∗∗∗ P < 0.0001 by two-way ANOVA, followed by Tukey's multiple comparisons test).

Article Snippet: Adherent cells were harvested at passage three for flow cytometry, where they were stained with fluorescence-labeled antibodies against Pax7 (NBP2-34706 PE, Novus, USA) and desmin (Y&M, BYK-1026R-FITC) ( ).

Techniques: Phospho-proteomics, Western Blot, Activation Assay, Control, Immunofluorescence, Staining, Immunoprecipitation, Plasmid Preparation, shRNA, Infection