cell culture mouse myogenic cell line c2c12  (ATCC)

Journal: iScience

Article Title: Trichinella spiralis inhibits myoblast differentiation by targeting SQSTM1/p62 with a secreted E3 ubiquitin ligase

doi: 10.1016/j.isci.2024.109102

Figure Lengend Snippet: Ts -RNF causes abnormal differentiation of myoblasts (A and B) Confocal microscopy images of tubulin and myotube heavy chain (MyHC) in myotubes eight days after initiation of differentiation. Ts -RNF (green), MyHC (red), and Hoechst (blue). Cells were transfected with a plasmid with gene encoding the Ts -RNF gene. Untransfected cells (Blank) and cells transfected with empty vector served as negative controls (NC). Representative single optical sections and merged images are shown. Scale bars: 20 μm. (C) The CCK-8 assay was used to analyze the viability of Ts -RNF- and NC-transfected cells. The expression levels of the Ki67 gene in the NC group and Ts -RNF group were quantitatively analyzed in undifferentiated C2C12 cells. Data are shown as the means ± SEMs from three independent experiments (n = 3). Statistical analysis was performed using independent samples t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant. (D) The ubiquitination level of myoblast differentiation was analyzed at different time points in the NC and Ts -RNF groups using an in vitro ubiquitination assay (DM differentiation medium). (E) Representative immunoblots of differentiation markers (MyoD, MyoG, MyHC) at different time points in the NC and Ts -RNF groups. (F) The mRNA expression levels of the fusion markers (Myomarker and Myomixer) at different time points in the NC group and Ts -RNF group. Data are shown as the means ± SEMs from three independent experiments (n = 3). Statistical analysis was performed using Mann-Whitney U test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant.

Article Snippet: Mouse C2C12 myogenic cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured at 37°C in 5% CO2 in growth medium (GM) consisting of high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, USA), 4 mM L-glutamine, 100 U/ml penicillin and 100 μg/mL streptomycin.

Techniques: Confocal Microscopy, Transfection, Plasmid Preparation, CCK-8 Assay, Expressing, Ubiquitin Proteomics, In Vitro, Western Blot, MANN-WHITNEY

Journal: iScience

Article Title: Trichinella spiralis inhibits myoblast differentiation by targeting SQSTM1/p62 with a secreted E3 ubiquitin ligase

doi: 10.1016/j.isci.2024.109102

Figure Lengend Snippet: Ts -RNF interacted with SQSTM1/p62 to influence mitochondrial clearance in C2C12 cells (A) Confocal microscopy images of p62 on day 8 of differentiation. p62 (red), Ts -RNF (green), and Hoechst (blue). Untransfected cells (Blank) and cells transfected with empty vector served as negative controls (NC). Representative single optical sections and merged images are shown. Scale bars: 20 μm. (B) Representative immunoblots of Pink1(PTEN induced putative kinase 1), Parkin (PARK2), P62(SQSTM1), and LC3B(MAP1LC3) at different time points in the NC and Ts -RNF groups. (C) Confocal microscopy images of mitochondrial membrane potential on the day 8 of differentiation. Ts -RNF (green), TMRE, tetramethylrhodamine ethyl ester (red), and Hoechst (blue). Untransfected cells (Blank) and cells transfected with empty vector served as controls (NC). Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (CCCP, 25 μM), an uncoupler of mitochondrial oxidative phosphorylation, was used as a control for mitochondrial depolarization. Representative single optical sections and merged images are shown. Scale bars: 50 μm. (D) Superoxide anion kit assay analyzed the superoxide anion production of Ts -RNF and NC group on day 8 of differentiation (OD: 450 nm). Superoxide dismutase (SOD) was used as a positive control. Data are shown as the means ± SEMs from three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant. (E) Transmission electron microscope (TEM) was used to analyze the structure of mitochondria and lysosomes on the day 8 of differentiation. Red frame: autolysosome (autolysosomes were identified as single-membrane structures containing cytoplasmic components at various stages of degradation), Green frame: mitochondrial (mitochondria were identified by their characteristics and cristae), Yellow frame: lipid droplets. Magnification: 5.0 k. Scale bar: 1 μm. (F) Western blotting was used to detect the total protein expression and phosphorylation levels of AKT (protein kinase B), MAPK (mitogen-activated protein kinase), mTOR (mammalian target of rapamycin), ULK1 (Unc-51-like autophagy activating kinase 1) at different time points in the NC and Ts -RNF groups. The data and images are representative of at least three independent experiments. The samples were derived from the same experiment, and the blots were processed in parallel.

Article Snippet: Mouse C2C12 myogenic cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured at 37°C in 5% CO2 in growth medium (GM) consisting of high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, USA), 4 mM L-glutamine, 100 U/ml penicillin and 100 μg/mL streptomycin.

Techniques: Confocal Microscopy, Transfection, Plasmid Preparation, Western Blot, Membrane, Phospho-proteomics, Control, Positive Control, Transmission Assay, Microscopy, Expressing, Derivative Assay

Journal: iScience

Article Title: Trichinella spiralis inhibits myoblast differentiation by targeting SQSTM1/p62 with a secreted E3 ubiquitin ligase

doi: 10.1016/j.isci.2024.109102

Figure Lengend Snippet:

Article Snippet: Mouse C2C12 myogenic cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured at 37°C in 5% CO2 in growth medium (GM) consisting of high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, USA), 4 mM L-glutamine, 100 U/ml penicillin and 100 μg/mL streptomycin.

Techniques: Recombinant, Ubiquitin Proteomics, Conjugation Assay, Transfection, Plasmid Preparation, Software