Journal: Journal of Biological Chemistry

Article Title: Proinflammatory Cytokine Tumor Necrosis Factor (TNF)-like Weak Inducer of Apoptosis (TWEAK) Suppresses Satellite Cell Self-renewal through Inversely Modulating Notch and NF-κB Signaling Pathways

doi: 10.1074/jbc.m113.517300

Figure Lengend Snippet: FIGURE 1. TWEAK inhibits self-renewal of satellite cells on cultured myofibers. Single myofiber cultures were established from extensor digitorum longus muscle of WT mice and treated with TWEAK (10 ng/ml). After 72 h, satellite cell-myoblast clusters (containing 4 cells) on single myofibers were labeled with antibodies against Pax7 and MyoD. Nuclei were counterstained with DAPI. Representative merged images of Pax7-, MyoD-, and DAPI-stained cells on myofi- bers in control (A) and TWEAK-treated cultures (B) are presented. The percentage of Pax7 cells (C) per cluster and MyoD cells (D) per cluster in untreated and TWEAK-treated myofiber cultures are indicated. Quantitative analysis of the percentage of self-renewing (Pax7/MyoD) (E), proliferating (Pax7/MyoD) (F), anddifferentiating(Pax7/MyoD)(G)satellitecells/myoblastsonmyofibersinuntreatedandTWEAK-treatedmyofibercultures.n 4ineachgroup.Errorbars represent S.D. *, p 0.05 values significantly different from untreated cultures.

Article Snippet: Suspended fibers were cultured in horse serum-coated 6-well plates in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 2% chicken embryo extract (Accurate Chemical, Westbury, NY), and 1% penicillin-streptomycin for 72 h. Cultured fibers were then fixed in 4% paraformaldehyde and stained for Pax7 (1:20, Developmental Studies Hybridoma Bank) and MyoD (1:200, Santa Cruz Biotechnology).

Techniques: Cell Culture, Labeling, Staining, Control

Journal: Journal of Biological Chemistry

Article Title: Proinflammatory Cytokine Tumor Necrosis Factor (TNF)-like Weak Inducer of Apoptosis (TWEAK) Suppresses Satellite Cell Self-renewal through Inversely Modulating Notch and NF-κB Signaling Pathways

doi: 10.1074/jbc.m113.517300

Figure Lengend Snippet: FIGURE 2. TWEAK represses Pax7 levels in cultured satellite cells. Primary satellite cells/myoblasts were isolated from WT mice. After purification, the cells weretreatedwith10ng/mlofTWEAKfor24h.A,representativephotomicrographsofsatellitecellsafterstainingwithantibodiesagainstPax7andMyoD.Nuclei were counterstained with DAPI. Scale bar: 50 m. B, percentage of Pax7 and MyoD cells in control and TWEAK-treated cultures. n 4 in each group. C, relative mRNA levels of Pax7 and MyoD in satellite cell cultures after 24 h of TWEAK (10 ng/ml) treatment. n 4 in each group. D, satellite cells were treated with 10 ng/ml of TWEAK for the indicated times followed by immunoblotting to detect protein levels of Pax7, MyoD, and tubulin. Representative immunoblots are presented here. E, densitometry quantification of the percentage decrease in Pax7 protein levels in response to TWEAK treatment in three independent experiments. Error bars represent S.D. *, p 0.05, values significantly different from untreated cells.

Article Snippet: Suspended fibers were cultured in horse serum-coated 6-well plates in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 2% chicken embryo extract (Accurate Chemical, Westbury, NY), and 1% penicillin-streptomycin for 72 h. Cultured fibers were then fixed in 4% paraformaldehyde and stained for Pax7 (1:20, Developmental Studies Hybridoma Bank) and MyoD (1:200, Santa Cruz Biotechnology).

Techniques: Cell Culture, Isolation, Purification, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Proinflammatory Cytokine Tumor Necrosis Factor (TNF)-like Weak Inducer of Apoptosis (TWEAK) Suppresses Satellite Cell Self-renewal through Inversely Modulating Notch and NF-κB Signaling Pathways

doi: 10.1074/jbc.m113.517300

Figure Lengend Snippet: FIGURE 4. Genetic ablation of TWEAK improves the number of Pax7 cells and Notch signaling in cardiotoxin-injected TA muscle of mice. TA muscle of 3-month-old WT and TWEAK-KO mice were injected with 100 l of cardiotoxin (CTX, 10 M) and 5 days later the muscle was isolated and processed for immunostaining for Pax7 and laminin. Nuclei were identified by counterstaining with DAPI. Representative photomicrographs presented here demonstrate increased immunostaining for Pax7 in CTX-injected TA muscles of TWEAK-KO mice compared with WT mice. B, quantification of the number of Pax7 cells per myofiber in TA muscle of WT (n 3) and TWEAK-KO mice (n 5) 5 days post-CTX injection. C, single myofiber cultures were prepared from WT and TWEAK-KO mice and immunostained for Pax7 and MyoD and the percentage of Pax7 cells per cluster was calculated (n 8 in each). D, relative mRNA levels of Pax7, Notch1, Notch2, Notch3, Jagged1, Dll1, Hes1, Heyl, and Hes6 in 5-day CTX-injected TA muscle of WT and TWEAK-KO mice measured by QRT-PCR assay. n 3–4 in each group. Error bars represent S.D. *, p 0.05, values significantly different from WT mice.

Article Snippet: Suspended fibers were cultured in horse serum-coated 6-well plates in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 2% chicken embryo extract (Accurate Chemical, Westbury, NY), and 1% penicillin-streptomycin for 72 h. Cultured fibers were then fixed in 4% paraformaldehyde and stained for Pax7 (1:20, Developmental Studies Hybridoma Bank) and MyoD (1:200, Santa Cruz Biotechnology).

Techniques: Injection, Isolation, Immunostaining, Muscles, Quantitative RT-PCR

Journal: Life science alliance

Article Title: STAT3 inhibition recovers regeneration of aged muscles by restoring autophagy in muscle stem cells.

doi: 10.26508/lsa.202302503

Figure Lengend Snippet: Figure 6. STAT3 inhibition induces autophagy by disrupting the STAT3/PKR complex and activating pro-autophagic gene transcription in vitro. C2C12 cell line was treated for 24 h with PBS (NT) or the STAT3i at the concentration of 50 μM, then harvested for analysis. (A) qRT–PCR for Myod and Socs3 normalized for Gapdh (n = 3 for MyoD and n = 4 for Socs3; values represent the mean ± s.d., **P < 0.01 and ***P < 0.001 by a t test). (B) qRT–PCR for Lc3, Atg9A, Ulk1, Atg7, Bnip3, Atg4, Becn1, and Bcl2 normalized for Gapdh (n = 3; values represent the mean ± s.d., *P < 0.05, **P < 0.01, and ***P < 0.001 by a t test). (C) Phospho-eIF2α levels in NT or STAT3i-treated

Article Snippet: The following antibodies were used: LC3B (#2775S; Cell Signaling), p62 (#P0067; Sigma-Aldrich), total STAT3 (#4904S; Cell Signaling), P-STAT3 (#4113S; Cell Signaling), MYOD (5.8; Novus Biologicals), phospho-eIF2α (#3398S; Cell Signaling), GAPDH (Santa Cruz), and vinculin (sc-73614; Santa Cruz).

Techniques: Inhibition, In Vitro, Concentration Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Circular Smad1-Encoded Polypeptide Regulates Myogenesis

doi: 10.64898/2026.02.26.708385

Figure Lengend Snippet: A. Representative images of myoblast and myotube in C2C12 cells. B. Bar chart showing levels of myogenic markers Myog and Myod1 in differentiating C2C12 cells, normalised to Gapdh mRNA by RT-qPCR. C. Representative immunoblot detecting bands of MYOG, MYOD1 and MYH3 in C2C12 MB, MT (left), and their relative levels in myotubes normalised to HSP90 loading control shown in the densitometry bar plot (right). D-E. Representative polysome profiling graphs of RNA in different fractions of C2C12 myoblasts (left) and myotubes (right), distinguishing the monosome (F1-F6) and polysome (F7-F12) fractions. F. Schematic diagram showing bioinformatic workflow used for the identification of circRNAs from RNA-seq read input. G. Venn diagram showing the number of common and exclusive circRNAs between myoblast, myotube and riboCIRC v1. H. Area plot showing the distribution of circRNA population depending on the number of exons or introns. I. Bar plot showing the grouping of circRNAs based on spliced length range. J. Pie chart showing the percentage of the identified circRNAs that originate from exonic (circRNAs) or intronic (ciRNAs) regions of the genome. Data displayed in panels B and C represent the mean ± SEM of at least 3 biological replicates, and * indicates significance with a p-value <0.05.

Article Snippet: Protein lysates of transfected myotubes were prepared in 1X RIPA buffer and assessed by immunoblotting with antibodies against muscle-specific markers, including MYOD1 (ABclonal), MYOG (Thermo Fisher Scientific), and MYH3 (SCBT).

Techniques: Quantitative RT-PCR, Western Blot, Control, RNA Sequencing

Journal: bioRxiv

Article Title: Circular Smad1-Encoded Polypeptide Regulates Myogenesis

doi: 10.64898/2026.02.26.708385

Figure Lengend Snippet: A. CircSmad1 contains a junction-spanning ORF and multiple m6A sites, which recruit polysomes and undergo cap-independent translation into a 194aa polypeptide called circSmad1-194aa. B. CircSmad1-194aa contains a conserved DNA-binding MH1 domain, which mediates its nuclear translocation and competitive binding at the Id1 -BRE promoter site, leading to downregulation of Id1 . C. Reduced expression of Id1 derepresses the activity of basic HLH myogenesis regulatory factors (Myod1 and Myogenin), inducing myogenesis in mouse C2C12 myoblasts.

Article Snippet: Protein lysates of transfected myotubes were prepared in 1X RIPA buffer and assessed by immunoblotting with antibodies against muscle-specific markers, including MYOD1 (ABclonal), MYOG (Thermo Fisher Scientific), and MYH3 (SCBT).

Techniques: Binding Assay, Translocation Assay, Expressing, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Coordinated regulation of the ribosome and proteasome by PRMT1 in the maintenance of neural stemness in cancer cells and neural stem cells

doi: 10.1016/j.jbc.2021.101275

Figure Lengend Snippet: Effect of MYOD1-induced differentiation on the expression of ribosome and proteasome components and epigenetic factors. A , phenotypic change in A375 cells infected with virus containing MYOD1. Cells infected with virus containing the empty vector were used as a control. B , IB detection of expression change of a series of proteins, as indicated, in control A375 cells and cells with forced MYOD1 expression. C , phenotypic change in A549 cells in response to MYOD1 expression. D , detection of protein expression, as indicated, in control cells and cells with MYOD1 expression using IB. E , IF detection of expression and subcellular distribution of a muscle cell marker, ribosome and proteasome proteins, and the proteasome 20S subunit in control cells and cells with MYOD1 expression. F , expression alteration in a series of proteins, as indicated, in wild-type C2C12 (C2C12 WT ) cells and the cells with knockout of Myod1 gene (C2C12 Myod1 −/− ).

Article Snippet: Primary antibodies were EZH2 (Cell Signaling Technology, #5246), HDAC1 (Cell Signaling Technology, #5356), LSD1 (Cell Signaling Technology, #2139), MAP2 (Abcam, #ab183830), MEF2C (Cell Signaling Technology, #5030), MYOD1 (Novus Biologicals, #NB100-56511), NESTIN (R&D systems, #AF2736), NEUROD1 (Cell Signaling Technology, #4373), NF-L (Cell Signaling Technology, #2837), PAX6 (Abcam, #ab195045), PRMT1 (Cell Signaling Technology, #2449), PSMA2 (Abclonal, #A2504), PSMD2 (Abclonal, #A1999), RPL26 (Abclonal, #A16680), RPS3 (Abclonal, #A2533), SOX1 (Abcam, #ab109290), SOX2 (Cell Signaling Technology, #23064), TUBB3 (Cell Signaling Technology, #5568), 20S α + β (Abcam, #ab22673).

Techniques: Expressing, Infection, Virus, Plasmid Preparation, Control, Marker, Knock-Out