mouse skeletal muscle myoblast cells  (ATCC)

Journal: bioRxiv

Article Title: Autophagy selectively clears ER in inflammation-induced muscle atrophy

doi: 10.1101/2025.05.06.650963

Figure Lengend Snippet: (A) Experimental workflow: C2C12 myoblasts were differentiated for seven days in light medium (K0 = Lysine 0 [ 12 C 6 , 14 N 2 ]; R0 = Arginine 0 [ 12 C 6 , 14 N 4 ]) into myotubes (= t0). Medium was then replaced with heavy medium (K8 = Lysine 8 [ 13 C 6 , 15 N 2 ]; R10 = Arginine 10 [ 13 C 6 , 15 N 4 ]) for 24 or 72 hours (t24, t72), with or without TNF-α to induce atrophy. Proteolytic inhibitors (BafA1: Bafilomycin A1 inhibiting autophagy; Lac: Lactacystin inhibiting the UPS) were administered for three hours prior to cell harvest, and cells were analyzed using LC-MS/MS. n = 3 replicates. (B) Protein clustering based on total intensity dynamics of non-inhibitor treated homeostatic and atrophying cells: Proteins were categorized into four clusters according to their normalized total intensity values (light + heavy) at t0, t24, and t72. Percentages of protein counts were rounded up. (C) Proteins of each cluster of total intensities were subjected to GO term enrichment (biological processes) analysis.

Article Snippet: C2C12 mouse skeletal muscle myoblasts (ATCC # CRL-1772) were grown adherently and undifferentiated in DMEM (Sigma Aldrich # D5796) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco # 10500-064) and 1% Pen/Strep (P/S) (Sigma Aldrich # P4333).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Autophagy selectively clears ER in inflammation-induced muscle atrophy

doi: 10.1101/2025.05.06.650963

Figure Lengend Snippet: OPP (O-propargyl-puromcyin) labeling of differentiated myotubes (homeostasis at 24 h (H24) and 72 h (H27)) and TNF-α-induced atrophying C2C12 cells (24 h (A24) and 72 h (A72)) was used to measure protein synthesis rates at time points t24 and t72. Cycloheximide (CHX) was employed for 90 min as a control to inhibit protein synthesis. Puncta were manually counted across 20 images per condition, each containing over 200 cells. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar: 60 µm.

Article Snippet: C2C12 mouse skeletal muscle myoblasts (ATCC # CRL-1772) were grown adherently and undifferentiated in DMEM (Sigma Aldrich # D5796) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco # 10500-064) and 1% Pen/Strep (P/S) (Sigma Aldrich # P4333).

Techniques: Labeling, Control

Journal: bioRxiv

Article Title: Autophagy selectively clears ER in inflammation-induced muscle atrophy

doi: 10.1101/2025.05.06.650963

Figure Lengend Snippet: (A-C) Flow cytometry-based assay measuring autophagic turnover by staining membrane-bound LC3-II with a fluorescently labeled antibody ( Alsaleh et al ., 2020 ) (A) Representative histograms display the shift in LC3-II signal following 3 h BafilomycinA1 (BafA1) treatment (B) Elevated LC3-II signal intensity (gMFI, geometric mean fluorescence intensity) in atrophying (A24, A72) cells compared to homeostatic cells (H24, H72) in dimethylsulfoxide (DMSO)-treated samples. N = 4 replicates. (C) Autophagic turnover was calculated by determining the difference in LC3-II signal intensity (gMFI) between BafA1-treated and DMSO-treated cells over a 3h incubation period. N = 4 replicates. (D) Accumulated proteins upon BafilomycinA1 (BafA1) in ANOVA comparisons of atrophying (A24+BafA1 and A72+BafA1) vs homeostatic (H24+BafA1 and H72+BafA1) C2C12 cells in the light channel with log 2 FC set > 0 and adj. p-value set at < 0.05. Numbers in Venn diagram depict amount of significantly regulated proteins in respective dataset comparison. Red dots shown in volcano plot of A72+BafA1 vs. H72+BafA1 depict annotated autophagic machinery components.

Article Snippet: C2C12 mouse skeletal muscle myoblasts (ATCC # CRL-1772) were grown adherently and undifferentiated in DMEM (Sigma Aldrich # D5796) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco # 10500-064) and 1% Pen/Strep (P/S) (Sigma Aldrich # P4333).

Techniques: Flow Cytometry, Staining, Membrane, Labeling, Fluorescence, Incubation, Comparison

Journal: bioRxiv

Article Title: Autophagy selectively clears ER in inflammation-induced muscle atrophy

doi: 10.1101/2025.05.06.650963

Figure Lengend Snippet: (A) Immunocytochemistry staining of C2C12 (atrophying) myotubes for p62/Sqstm1 and myosin heavy chain (MyHC) highlighting the M band region of the sarcomere. (B) Corresponding line plots display fluorescence intensity profiles for each channel along the length of the sarcomere. Scale bar 20 µm. (C) Accumulated myofibrillar proteins upon BafilomycinA1 (BafA1) and Lactacystin (Lac) treatment in ANOVA comparisons of homeostatic (H) and atrophying (A) C2C12 cells in the light channel with log 2 FC set at > 0 and adj. p-value set at < 0.05. White: not significantly accumulated; grey: not significant.

Article Snippet: C2C12 mouse skeletal muscle myoblasts (ATCC # CRL-1772) were grown adherently and undifferentiated in DMEM (Sigma Aldrich # D5796) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco # 10500-064) and 1% Pen/Strep (P/S) (Sigma Aldrich # P4333).

Techniques: Immunocytochemistry, Staining, Fluorescence

Journal: bioRxiv

Article Title: miR-29a-3p, a new myokine orchestrating resistance exercise via coordinated metabolic responses

doi: 10.1101/2024.05.14.592416

Figure Lengend Snippet: (A) Schematic representation of electrostimulation protocol on C2C12 cells and isolation of miRNAs from EVs and cells. (B-D) Relative expression levels of miR-30a-5p (B), let-7f-5p (C), and miR-29a-3p (D) in cells and EVs at 0, 3, and 20 hours after electrostimulation in non-electrostimulated (C, n=4) and electrostimulated (EPS, n=4) cells. (E-H) Relative expression of miR-29 family members (E), cluster 1 miRNAs (F), cluster 2 miRNAs (G), and miR-143-3p (H) in C2C12 myotubes where miR-29a-3p expression was inhibited by 70% (miR-29a-3p dKD, n=4) and in control cells (NC, n=4). (I and J) Relative expression of miR-30a-5p and miR-143-3p in non-electrostimulated (C, n=4) and electrostimulated (EPS, n=4) miR-29a-3p dKD cells at 0, 3, and 20 hours after electrostimulation. Data are represented as mean ± SEM. Each dot represents the mean technical duplicate of each cell replicate. Statistical significance was determined using Student’s t-test for independent samples.

Article Snippet: The mouse skeletal muscle myoblast cell line C2C12 were obtained from the American Type Culture Collection (ATCC CRL-1772).

Techniques: Isolation, Expressing, Control

Journal: Nutrients

Article Title: New Trends to Treat Muscular Atrophy: A Systematic Review of Epicatechin

doi: 10.3390/nu16020326

Figure Lengend Snippet: Summary of the main EC supplementation parameters–Studies in culture cells.

Article Snippet: Edwards et al., 2022 [ ] , Epi: #E1753 Sigma , Mouse skeletal muscle C2C12 myoblasts , To investigate the effects of EC and HA (hippuric acid) on skeletal muscle morphology and metabolism investigating an in vitro model of muscle atrophy with dexamethasone. , Divided into 6 groups: VC-CTRL: vehicle control. VC-DEX: cells incubated in dexamethasone; EPI-CTL: cells incubated with 25 μM EC; EPI + DEX: cells were incubated in 25 μM EC and 100 μM DEX; HA-CTL: cells incubated in 25 μM HA.; HA + DEX: cells incubated with 25 μM HA and 100 μM DEX , 25 μM EPI and 100 μM DEX. , 24h of treatment protocol , Cells were incubated in DMEM (5mM glucose), followed by 6 days of differentiation, and received 24 h of treatment. , PGC1 α, ACC, and TFAM (regulators of mitochondrial function) were significantly lower in DEX-treated versus CTL cells (Control). However, EPI or HA partially attenuated the proteolysis in DEX-treated groups by preserving the expression of LC3 and caspase-3 protein. Myotube diameter was significantly greater in EPI-DEX and HA- DEX. p ≤ 0.05.

Techniques: Negative Control, Incubation, Positive Control, Cell Culture, Differentiation Assay, Cell Differentiation, Expressing, In Vitro, Preserving