Journal: Frontiers in Genetics

Article Title: RNA-seq analysis reveals a positive role for NGF in the myogenic differentiation of bovine skeletal muscle satellite cells

doi: 10.3389/fgene.2025.1713817

Figure Lengend Snippet: Role of NGF in the regulation of myogenic differentiation. (A) Determination of the mRNA levels of Pax7, a marker gene of proliferation. (B) Determination of the protein levels of Pax7. (C) Detection of the effect of the over-NGF on the proliferation of bSMSCs using the CCK8 method. (D) Detection of the effect of the si-NGF on the proliferation of bSMSCs using the CCK8 method. (E) Determination of the mRNA expression of MyHC, MyoG and MyoD1-myogenic differentiation marker genes. (F) Determination of the protein expression of MyHC, MyoG and MyoD1. ** p < 0.01; *** p < 0.001.

Article Snippet: Primary antibodies against NGF(Bioss, bs-10806R, Beijing, China, 1:2000 dilution), PAX7 (DSHB, Pax7, United States, 1:1500 dilution), MyoD1(Bioss, bs-62914R, Beijing, China, 1:1500 dilution), MyHC(DSHB,10F5, United States, 1:1500 dilution), MyoG (Bioss, bs-3550R, Beijing, China, 1:1500 dilution), Akt (CST, 9272S, United States, 1:2000 dilution), p-Akt (CST, 9271S, United States,1:2000 dilution), PI3K(CST, 4249S, United States, 1: 2000 dilution), p-PI3K(CST, 4228S, United States, 1: 2000 dilution)and GAPDH(Proteintech, 60004-1-Ig, United States, 1: 5000 dilution) were diluted with TBST, and the total protein concentrations of the samples were determined using a BCA assay.

Techniques: Cell Characterization, Marker, Expressing

Journal: eLife

Article Title: Branched actin polymerization drives invasive protrusion formation to promote myoblast fusion during mouse skeletal muscle regeneration

doi: 10.7554/eLife.103550

Figure Lengend Snippet: ( A ) Schematic diagram of tamoxifen and BaCI 2 treatment and subsequent cell proliferation and differentiation analyses at dpi 2.5 and 4, respectively. ( B ) Immunostaining with anti-Laminin, anti-Pax7, and anti-Ki67 of cross-sections of TA muscles from Ctrl and mutant mice at dpi 2.5. The boxed areas are shown on the right. Scale bar: 30 µm. ( C ) Immunostaining with anti-Laminin and anti-MyoG of cross-sections of TA muscles from Ctrl and mutant mice at dpi 4. The boxed areas are shown at the top-right corner. Scale bar: 30 µm. ( D ) Quantification of the percentage of proliferating satellite cells (% of Ki67 + cells in the Pax7 + cells) in TA muscles from Ctrl and mutant mice of the indicated genotypes. n=3 mice of each genotype were examined. Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed Student’s t -test. ns: not significant. ( E ) Quantification of the percentage of MyoG + nuclei in the total cells in TA muscles from Ctrl and mutant mice of the indicated genotypes. n=3 mice of each genotype were examined. Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed Student’s t -test. ns: not significant. ( F ) Western blot analysis for MymX and MymK in TA muscles from Ctrl and mutant mice of the indicated genotypes at dpi 4. One sample of each Ctrl and mutant genotype is shown. ( G, H ) Quantification of MymX and MymK protein expression in the Ctrl and littermate mutant mice as shown in ( F ). The band intensity of each protein was normalized against β-tubulin. The y axis indicates the expression of MymX or MymK in different mutants relative to the control mice. n=3 mice of each genotype were examined. Mean ± s.d. values are shown in the bar graphs, and significance was determined by two-tailed Student’s t -test. *p<0.05; ns: not significant. Figure 3—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Then, the sections were fixed in 2% PFA for 5 minutes at RT, washed three times with PBS, and incubated with blocking buffer supplemented with M.O.M blocking reagent (1:25; Vector; MKB-2213-1) for 60 minutes at RT, followed by overnight incubation with mouse anti-Pax7 (1:2; DSHB; Pax7), mouse anti-MyoG (1:2; DSHB; F5D), rat anti-Laminin-2 (1:500; Sigma; L0663), and rat anti-Ki67 (1:500; Thermo Fisher Scientific; 14-5698-82) at 4°C.

Techniques: Immunostaining, Muscles, Mutagenesis, Two Tailed Test, Western Blot, Expressing, Control