pax7 ab 528428 myod mouse igg2b  (Developmental Studies Hybridoma Bank)

Journal: Nature Communications

Article Title: Reconstructing epigenomic dynamics through a single-cell multi-epigenome data integration framework

doi: 10.1038/s41467-025-67016-9

Figure Lengend Snippet: a Schematic overview of data integration for multiple sci-mtChIL-seq datasets. RNAPII fragments were counted at the gene level and used as anchors to embed all datasets into a shared low-dimensional space. b Diagram of the skeletal muscle differentiation system using NIH3T3 TetOn3G/MyoD cells. c UMAP visualization of RNAPII counts at the single-cell level. Cells are colored by (left) sampling time point, (middle) paired epigenomic target, and (right) identified cluster. d Cell composition within each cluster. (Top) Distribution by sampling time. (Bottom) Distribution by paired target. e Heatmap of RNAPII accumulation across differentially expressed genes (DEGs). For each cluster, the top 200 cells (ranked by RNAPII count) and the top 50 DEGs (ranked by variance) are shown. f UMAP visualization of RNAPII accumulation levels per cell for representative DEGs. The values indicate niormalized counts of RNAPII for each coding region. g Gene Ontology (GO) enrichment analysis results for each cluster, based on the top 300 DEGs per cluster. h Heatmap showing mean accumulation levels of each epigenomic factor across clusters. i UMAP visualization of the accumulation levels per cell of epigenomic factors for representative DEGs.

Article Snippet: NIH3T3 Tet-On 3G/pCW-MyoD-Blast cells were cultured in DMEM supplemented with 10% Tet-Approved FBS (Takara; 631101).

Techniques: Sampling

Journal: The Journal of Biological Chemistry

Article Title: A-to-I RNA editing impairs miR-376b-3p repression of RYBP in skeletal muscle satellite cells

doi: 10.1016/j.jbc.2025.111006

Figure Lengend Snippet: Effects of WT and edited miR-376b-3p on the differentiation of goat MuSCs . A , expression of miR-WT and miR-E following transfection with miR-A, miR-G, or miR-NC in MuSCs, and the data were evaluated using the one-way ANOVA. Data are represented as the mean ± SD, n = 3 independent experiments. B , mRNA expression of differentiation markers MyoD (an early myogenic determination factor), MyoG (a marker of myoblast differentiation), and MyHC (indicative of terminal differentiation and myotube formation) following transfection with miR-A, miR-G, or miR-NC in MuSCs. Data were analyzed using one-way ANOVA and are presented as the mean ± SD (n = 3 independent experiments). C and D , Western blot analysis of MyoG, MyoD, and MyHC protein levels in MuSCs transfected with miR-A, miR-G, or miR-NC, and the data were evaluated using the one-way ANOVA. Data are represented as the mean ± SD, n = 2 independent experiments. E , immunofluorescence staining of MyHC to evaluate the differentiation potential of MuSCs after transfection with miR-A, miR-G, or miR-NC. The fusion index is quantified on the right , and the data were evaluated using the one-way ANOVA. Data are represented as the mean ± SD, n = 6 independent experiments. Asterisks indicate statistical significance: ∗ for p < 0.05, ∗∗ for p < 0.01, and “ns” for nonsignificant differences. MuSC, skeletal muscle satellite cell.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with the appropriate primary antibodies Pax7 (1:200 dilution; Santa Cruz), PCNA (1:1000 dilution; Abclonal), MyoG (1:1000 dilution; Bioss), MyoD (1:1000 dilution; Proteintech), MyHC (1:1000 dilution; Zen Bio), and β-tublin (1:2000 dilution; Zen Bio), followed by incubation with horseradish peroxidase–conjugated secondary antibodies (1:5000 dilution; Zen Bio) at 37 °C for 2 h. Protein signals were visualized using enhanced chemiluminescence reagents (Pierce). β-tubulin was used as a loading control.

Techniques: Expressing, Transfection, Marker, Western Blot, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: A-to-I RNA editing impairs miR-376b-3p repression of RYBP in skeletal muscle satellite cells

doi: 10.1016/j.jbc.2025.111006

Figure Lengend Snippet: Effects of miR-376b-3p and its edited form and RYBP overexpression on MuSC differentiation . A , quantification of cotransfection efficiency of RYBP , miR-WT, and miR-E at the differentiation stage of MuSCs, and the data were evaluated using the one-way ANOVA. Data are represented as the mean ± SD, n = 3 independent experiments. B , mRNA expression of myogenic differentiation markers following cotransfection of pRYBP with miR-A or miR-G, and the data were evaluated using the one-way ANOVA. Data are represented as the mean ± SD, n = 3 independent experiments. C and D , Western blot analysis of MyoG, MyoD, and MyHC protein levels in MuSCs after cotransfection with pRYBP and miR-A or miR-G, and the data were evaluated using the one-way ANOVA. Data are represented as the mean ± SD, n = 2 independent experiments. E , immunofluorescence staining of MyHC to assess myotube formation after cotransfection with pRYBP and miR-A or miR-G. The fusion index is shown on the right , and the data were evaluated using the one-way ANOVA. Data are represented as the mean ± SD, n = 6 independent experiments. Asterisks indicate statistical significance: ∗ for p < 0.05, ∗∗ for p < 0.01, and “ns” for nonsignificant differences. MuSC, skeletal muscle satellite cell; pRYBP, RYBP plasmid; RYBP, Ring1 and YY1 binding protein.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with the appropriate primary antibodies Pax7 (1:200 dilution; Santa Cruz), PCNA (1:1000 dilution; Abclonal), MyoG (1:1000 dilution; Bioss), MyoD (1:1000 dilution; Proteintech), MyHC (1:1000 dilution; Zen Bio), and β-tublin (1:2000 dilution; Zen Bio), followed by incubation with horseradish peroxidase–conjugated secondary antibodies (1:5000 dilution; Zen Bio) at 37 °C for 2 h. Protein signals were visualized using enhanced chemiluminescence reagents (Pierce). β-tubulin was used as a loading control.

Techniques: Over Expression, Cotransfection, Expressing, Cell Characterization, Western Blot, Immunofluorescence, Staining, Plasmid Preparation, Binding Assay

Journal: Cell Death & Disease

Article Title: TRPV2 in muscle satellite cells is crucial for skeletal muscle remodelling

doi: 10.1038/s41419-025-08242-3

Figure Lengend Snippet: a Timeline for TRPV2-deficiency induction, cell isolation, and staining. b Representative images of proliferating satellite cells derived from myofibres isolated from the EDL muscle of floxed and TRPV2 cKO mice after 4 days in culture. c Representative EdU incorporation assay (green) in MuSCs located c on myofibres and d around myofibres. TRPV2 (red) and DAPI (blue) staining were performed simultaneously. d Representative immunofluorescence staining of EdU (green), TRPV2 (red), and DAPI (blue) in MuSCs derived from myofibres isolated from the EDL muscle of floxed and TRPV2 cKO mice after 4 days in culture. e Ratio of EdU + cells. f Representative immunofluorescence staining of TRPV2 (green), Ki67 (red), and DAPI (blue) in MuSCs derived from myofibres isolated from the EDL muscle of floxed and TRPV2 cKO mice after 4 days in culture. g Ratio of Ki67 + cells. h Representative immunofluorescence staining of TRPV2 (green), MyoD (red), and DAPI (blue) in MuSCs derived from myofibres isolated from the EDL muscle of floxed and TRPV2-Pax7 -cKO mice after 4 days in culture. i Quantification of TRPV2 + cells per fibre. j Ratio of MyoD + cells among TRPV2 + cells. k Representative immunoblot showing PI3K, Akt, and phosphorylated Akt (P-Akt) expression in cultured MuSCs from floxed and cKO mice, with or without tamoxifen treatment. Data are presented as mean ± s.e.m. * P < 0.05 between the indicated groups (Student’s t -test for e , g , i , j ). # P < 0.05 (Tukey–Kramer test for k ). Scale bar, 100 µm.

Article Snippet: The following antibodies were used for immunostaining and immunoblotting analyses: anti-TRPV2 (Sigma; HPA044993, 1:1000 dilution), anti-Pax7 (Abcam; ab34360, 1:1000 dilution), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam; EPR16891 , 1:1000 dilution), anti-laminin (Abcam; ab11576, 1:1000 dilution), Akt (Cell Signaling Technology; 9272, 1:1000 dilution), P-Akt (Cell Signaling Technology; 4060, 1:1000 dilution), PI3K (Cell Signaling Technology; 4249, 1:1000 dilution), myoD (DSHB; D7F2-c, 1:1000 dilution), and anti-Ki67 (Proteintech; 28074-1-AP, 1:600 dilution).

Techniques: Cell Isolation, Staining, Derivative Assay, Isolation, Immunofluorescence, Western Blot, Expressing, Cell Culture