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

Journal: Frontiers in Veterinary Science

Article Title: miR-379-5p retards the proliferation and differentiation of goat skeletal muscle satellite cells by targeting LIN28B

doi: 10.3389/fvets.2025.1694160

Figure Lengend Snippet: miR-379-5p inhibits differentiation and promotes mitochondrial function in differentiating goat MuSCs. (A) Relative expression level of miR-379-5p in goat MuSCs transfected with NC or miR-379-5p mimics during the differentiation phase. (B) Relative mRNA levels of differentiation marker genes ( MyoD , MyoG , MyHC , Myf5 , and MEF2C ) in MuSCs transfected with NC or miR-379-5p mimics. (C) Western blot analysis of MyoD protein levels in differentiating MuSCs transfected with NC or miR-379-5p mimics. β-Tubulin was used as a loading control. (D) Representative immunofluorescence images of MyHC and DAPI in differentiating goat MuSCs after miR-379-5p overexpression. The right panel shows quantification of the fusion index. Scale bar = 50 μm. (E) Relative mRNA levels of mitochondrial marker genes ( CYCS , ATP5a1 , TFAM , and COX1 ) in differentiating MuSCs transfected with NC or miR-379-5p mimics. (F) Representative fluorescence images and quantification of mitochondrial membrane potential and ROS levels in differentiating MuSCs. Left: JC-1 staining reflects mitochondrial membrane potential. ROS levels were indicated by green fluorescence. Middle: Ratio of fluorescence intensity. Right: Quantification of ROS fluorescence intensity. Scale bar = 500 μm. Each experiment contained three biological replicates. * p < 0.05, ** p < 0.01.

Article Snippet: Primary antibodies included Pax7 (1:200, Santa Cruz Biotechnology, China), PCNA (1:200, Santa Cruz Biotechnology, China), β -Tubulin mouse mAb (1:5000, ZenBioScience, China), and MyoD polyclonal antibody (1:1000, Proteintech, United States).

Techniques: Expressing, Transfection, Marker, Western Blot, Control, Immunofluorescence, Over Expression, Fluorescence, Membrane, Staining

Journal: Frontiers in Veterinary Science

Article Title: miR-379-5p retards the proliferation and differentiation of goat skeletal muscle satellite cells by targeting LIN28B

doi: 10.3389/fvets.2025.1694160

Figure Lengend Snippet: LIN28B promotes differentiation and impairs mitochondrial function in differentiating goat MuSCs. (A) Relative mRNA level of LIN28B in goat MuSCs transfected with pCtrl or pLIN28B during the differentiation phase. (B) Relative mRNA levels of differentiation marker genes ( MyoD , MyoG , MyHC , MEF2C , and Myf5 ) in MuSCs transfected with pCtrl or pLIN28B. (C) Western blot analysis of MyoD protein levels in differentiating MuSCs transfected with pCtrl or pLIN28B. β-Tubulin was used as a loading control. (D) Representative immunofluorescence images of MyHC and DAPI in differentiating MuSCs transfected with pCtrl or pLIN28B. The right panel shows quantification of the fusion index. Scale bar = 100 μm. (E) Relative mRNA levels of mitochondrial marker genes ( CYCS , ATP5a1 , TFAM , COX1, and COX2 ) in differentiating MuSCs transfected with pCtrl or pLIN28B. (F) Representative fluorescence images and quantification of mitochondrial membrane potential and ROS levels in differentiating MuSCs. Left: JC-1 staining reflects mitochondrial membrane potential. ROS levels were indicated by green fluorescence. Middle: Ratio of fluorescence intensity. Right: Quantification of ROS fluorescence intensity. Scale bar = 500 μm. Each experiment contained three biological replicates. * p < 0.05, ** p < 0.01.

Article Snippet: Primary antibodies included Pax7 (1:200, Santa Cruz Biotechnology, China), PCNA (1:200, Santa Cruz Biotechnology, China), β -Tubulin mouse mAb (1:5000, ZenBioScience, China), and MyoD polyclonal antibody (1:1000, Proteintech, United States).

Techniques: Transfection, Marker, Western Blot, Control, Immunofluorescence, Fluorescence, Membrane, Staining

Journal: Frontiers in Veterinary Science

Article Title: miR-379-5p retards the proliferation and differentiation of goat skeletal muscle satellite cells by targeting LIN28B

doi: 10.3389/fvets.2025.1694160

Figure Lengend Snippet: miR-379-5p inhibits goat MuSC differentiation and improves mitochondrial function by targeting LIN28B . (A) Relative mRNA levels of MyoD , MyoG , and MyHC in goat MuSCs transfected with NC or miR-379-5p mimics in combination with pCtrl or pLIN28B during the differentiation phase. (B) Western blot analysis of MyoD protein levels in differentiating MuSCs transfected with NC or miR-379-5p mimics in combination with pCtrl or pLIN28B. β-Tubulin was used as a loading control. (C) Immunofluorescence staining of MyHC and analysis of myotube fusion index in differentiating goat MuSCs. Scale bar = 100 μm. (D) Relative mRNA levels of mitochondrial marker genes ( TFAM and COX1 ) in differentiating MuSCs transfected with NC or miR-379-5p mimics in combination with pCtrl or pLIN28B. (E) Representative fluorescence images illustrating mitochondrial membrane potential and ROS levels in differentiating MuSCs. Left: JC-1 staining indicates mitochondrial membrane potential. ROS production is indicated by green fluorescence. Middle: Ratio of fluorescence intensity. Right: Quantification of ROS fluorescence intensity. Scale bar = 500 μm. Each experiment contained three biological replicates. * p < 0.05, ** p < 0.01.

Article Snippet: Primary antibodies included Pax7 (1:200, Santa Cruz Biotechnology, China), PCNA (1:200, Santa Cruz Biotechnology, China), β -Tubulin mouse mAb (1:5000, ZenBioScience, China), and MyoD polyclonal antibody (1:1000, Proteintech, United States).

Techniques: Transfection, Western Blot, Control, Immunofluorescence, Staining, Marker, Fluorescence, Membrane