wt c2c12 mouse skeletal muscle myoblasts  (ATCC)

Journal: Autophagy Reports

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

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: Dynamic SILAC reveals distinct sets of proteins affected in their turnover upon TNF-α-induced muscle atrophy in C2C12 cells. (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 h (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 3 h prior to cell harvest, and cells were analyzed using LC-MS/MS. n = 3 replicates. (B) Myotube diameter was measured in differentiated C2C12 myotubes under homeostatic conditions at 24 h (H24; day 8 of differentiation) and 72 h (H72; day 10), as well as following TNF-α-induced atrophy at corresponding time points (A24 and A72). Measurements were performed using ImageJ on 209 individual myotubes per condition, quantified from 15-20 randomly selected images. Statistical significance was determined using ordinary one-way ANOVA with multiple comparisons (GraphPad Prism). Images used were acquired from OPP-stained samples from . (C) 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. (D) Proteins of each cluster of total intensities were subjected to GO term enrichment (biological processes) analysis.

Article Snippet: WT C2C12 mouse skeletal muscle myoblasts (ATCC, CRL-1772) and ssGFP-KDEL C2C12 cells (kindly provided and originally described by Buonomo et al . [ ]) 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: Multiplex sample analysis, Liquid Chromatography with Mass Spectroscopy, Staining

Journal: Autophagy Reports

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

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: TNF-α-induced atrophy leads to acute translation inhibition. (A) OPP (O-propargyl-puromcyin) labeling of differentiated myotubes (homeostatic 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. Arrows indicate puncta representing newly translated proteins. Asterisks indicate nuclei in differentiated, multinucleated myotubes. (B) 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. (C) Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar: 60 µm.

Article Snippet: WT C2C12 mouse skeletal muscle myoblasts (ATCC, CRL-1772) and ssGFP-KDEL C2C12 cells (kindly provided and originally described by Buonomo et al . [ ]) 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: Inhibition, Labeling, Control

Journal: Autophagy Reports

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

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: Increased autophagic turnover in TNF-α-induced atrophying C2C12 cells. (A) Western blot analysis of LC3B-II and p62 in C2C12 myotubes under homeostatic (H24, H72) and atrophic (A24, A72) conditions, with and without Bafilomycin (BafA1) treatment at time points 24 h and 72 h. Vehicle-treated (DMSO) cells serve as control. (B) LC3B-II and p62 intensities were normalized against Vinculin. n = 3 replicates. ns = not significant. (C-E) Flow cytometry-based assay measuring autophagic turnover by staining membrane-bound LC3-II with a fluorescently labeled antibody (C) Representative histograms display the shift in LC3-II signal following 3 h BafilomycinA1 (BafA1) treatment (D) 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. ns = not significant. (E) Autophagic turnover was calculated by determining the difference in LC3-II signal intensity (gMFI) between BafA1-treated and DMSO-treated cells over a 3 h incubation period. n = 4 replicates. (F) Accumulated proteins upon BafilomycinA1 (BafA1) treatment in ANOVA comparisons of atrophic (A24, A72) vs. homeostatic conditions (H24, H72) in the light channel (log 2 FC > 0 adj. p -value < 0.05). Left: Venn diagram numbers depict significantly regulated proteins per comparison. Right: Red dots in volcano plot depict autophagy machinery components.

Article Snippet: WT C2C12 mouse skeletal muscle myoblasts (ATCC, CRL-1772) and ssGFP-KDEL C2C12 cells (kindly provided and originally described by Buonomo et al . [ ]) 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: Western Blot, Control, Flow Cytometry, Staining, Membrane, Labeling, Fluorescence, Incubation, Comparison

Journal: Autophagy Reports

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

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: During TNF-a-induced atrophy, canonical autophagy does not mediate myofibrillar protein degradation, whereas ER-phagy emerges as the predominant form of selective autophagy. (A) Immunocytochemical staining of C2C12 (atrophying) myotubes for p62/Sqstm1 and myosin heavy chain (MyHC) highlighting the M band region of the sarcomere with corresponding line plots displaying fluorescence intensity profiles for each channel along the length of the sarcomere. Scale bar 20 µm. (B) Accumulated myofibrillar proteins upon BafilomycinA1 (+B) and Lactacystin (+L) treatment in ANOVA comparisons of homeostatic (H) and atrophying (A) C2C12 cells (light channel; log 2 FC > 0, adj. p -value < 0.05). White: not significantly accumulated; grey: not significant. (C) ANOVA of differentially regulated proteins (log 2 FC > 0; adj. p -value < 0.05). Left: Newly synthesized proteins in atrophy (A24 vs. H24 and A72 vs. H72) and accumulating (Light channel) selective autophagy receptors ± BafilomycinA1 (+B) treatment in atrophy (A24 vs. H24 and A72 vs. H72). Right: Accumulating ER components ± BafilomycinA1 (+B in atrophy (A24 vs. H24 and A72 vs. H24). White: not significantly accumulated; gray: not significant.

Article Snippet: WT C2C12 mouse skeletal muscle myoblasts (ATCC, CRL-1772) and ssGFP-KDEL C2C12 cells (kindly provided and originally described by Buonomo et al . [ ]) 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: Staining, Fluorescence, Synthesized

Journal: Autophagy Reports

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

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: Schematic model of proteostatic remodeling in TNF-α-induced muscle atrophy over time (24 h to 72 h): In C2C12 myotubes, TNF-α triggers selective remodeling of proteostasis, translation (Ribosomal and mitochondrial ribosomal proteins), ER stress, autophagic flux and autophagic cargo. Arrows represent changes observed under TNF-α-induced atrophy relative to the corresponding baseline control.

Article Snippet: WT C2C12 mouse skeletal muscle myoblasts (ATCC, CRL-1772) and ssGFP-KDEL C2C12 cells (kindly provided and originally described by Buonomo et al . [ ]) 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: Control

Journal: Preventive Nutrition and Food Science

Article Title: Puerarin-Rich Pueraria lobata Extract Attenuates H 2 O 2 - and Dexamethasone-Induced Atrophy in C2C12 Myoblasts and Myotubes

doi: 10.3746/pnf.2025.307

Figure Lengend Snippet: Cytotoxicity of Pueraria lobata extract powder (PEP) and its protective effect against H 2 O 2 -induced oxidative stress in C2C12 myoblasts. Cell viability was measured using an MTT assay. (A) C2C12 myoblasts were treated with the indicated concentrations of PEP for 24 h. Data are presented as a percentage of the untreated control group. (B) Cells were pretreated with PEP for 1 h, followed by cotreatment with 100 µM H 2 O 2 for a further 24 h. Data are presented as a percentage of the H 2 O 2 -only treated group. All values are presented as the mean±standard error of the mean (n=4). *** P <0.001 vs. the untreated normal control group; # P <0.05 vs. the H 2 O 2 -only treated group.

Article Snippet: Mouse skeletal muscle C2C12 myoblasts (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: MTT Assay, Control

Journal: Preventive Nutrition and Food Science

Article Title: Puerarin-Rich Pueraria lobata Extract Attenuates H 2 O 2 - and Dexamethasone-Induced Atrophy in C2C12 Myoblasts and Myotubes

doi: 10.3746/pnf.2025.307

Figure Lengend Snippet: Pueraria lobata extract powder (PEP) inhibits H 2 O 2 -induced apoptosis in C2C12 myoblasts. Cells were pretreated with the indicated concentrations of PEP for 1 h, followed by cotreatment with 100 µM H 2 O 2 for a further 24 h. (A) Apoptosis was quantified by measuring DNA fragmentation using a commercial ELISA. Data are presented as a percentage of the H 2 O 2 -only treated group. (B) Representative fluorescence microscopy images of cells stained with Hoechst 33258, showing nuclear morphology. Apoptotic nuclei appear brightly stained and condensed (magnification, ×200). All values are presented as the mean±standard error of the mean (n=6). *** P <0.001 vs. the untreated normal control group (CON); ## P <0.01 and ### P <0.001 vs. the H 2 O 2 -only treated group.

Article Snippet: Mouse skeletal muscle C2C12 myoblasts (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Enzyme-linked Immunosorbent Assay, Fluorescence, Microscopy, Staining, Control

Journal: Preventive Nutrition and Food Science

Article Title: Puerarin-Rich Pueraria lobata Extract Attenuates H 2 O 2 - and Dexamethasone-Induced Atrophy in C2C12 Myoblasts and Myotubes

doi: 10.3746/pnf.2025.307

Figure Lengend Snippet: Pueraria lobata extract powder (PEP) ameliorates dexamethasone (DEX)-induced atrophy in C2C12 myotubes. C2C12 myotubes were treated with 5 µM DEX in the presence or absence of the indicated concentrations of PEP for 24 h. (A) Cell viability was measured using an MTT assay and is expressed as a percentage relative to the control group. (B) Myotube diameter was quantified from immunofluorescence images and is expressed in micrometers (µm). (C) Representative immunofluorescence images of myotubes stained for myosin heavy chain (red) and nuclei with DAPI (blue). Scale bar=100 µm (magnification ×200). Data are presented as the mean±standard error of the mean (n=3). ** P <0.01 and *** P <0.001 vs. the control group (CON); # P <0.05, ## P <0.01, and ### P <0.001 vs. the DEX-only treated group.

Article Snippet: Mouse skeletal muscle C2C12 myoblasts (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: MTT Assay, Control, Immunofluorescence, Staining

Journal: The FASEB Journal

Article Title: MuRF1 Partners With TRIM72 to Impair Insulin Signaling in Skeletal Muscle Cells

doi: 10.1096/fj.202502066RR

Figure Lengend Snippet: MuRF1 interacts with TRIM72 in skeletal muscle cells. (A) Verification of MuRF1 interaction with endogenous TRIM72 in L6 myotubes that stably expressed GFP‐MuRF1. Total lysates (1 mg) from L6 myotubes that stably expressed GFP‐MuRF1 or GFP‐empty were subjected to GFP pulldown using GFP‐Trap agarose beads. The pulldown products were immunoblotted with the indicated antibodies. To confirm protein abundance and loading control, total lysates (lower panel) were immunoblotted with the indicated antibodies. Immunoblots were representative of three independent experiments. (B) Verification of endogenous MuRF1 interaction with endogenous TRIM72 in GFP‐MuRF1 knock‐in C2C12 myotubes. Total lysates (1 mg) from GFP‐MuRF1 knock‐in C2C12 myotubes were subjected to GFP pulldown using GFP‐Trap agarose beads or beads bound with IgG (as a control). The pulldown products and immunoprecipitates were immunoblotted with the indicated antibodies. To confirm protein abundance and loading control, total lysates (lower panel) were immunoblotted with the indicated antibodies. Immunoblots were representative of three independent experiments. (C) Verification of MuRF1 and TRIM72 interaction in HEK293 cells. HEK293 cells were transiently co‐transfected with FLAG‐TRIM72 and either with GFP‐MuRF1 or GFP‐empty (as a control) for 48 h. Total lysates (0.5 mg) were subjected to pulldown using GFP‐Trap agarose beads. The pulldown products were immunoblotted with the indicated antibodies. To confirm exogenous protein abundance, total lysates (lower panel) were immunoblotted with the indicated antibodies. Immunoblots were representative of one experiment.

Article Snippet: L6 rat and C2C12 mouse skeletal muscle myoblasts were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Stable Transfection, Quantitative Proteomics, Control, Western Blot, Knock-In, Transfection

Journal: The FASEB Journal

Article Title: MuRF1 Partners With TRIM72 to Impair Insulin Signaling in Skeletal Muscle Cells

doi: 10.1096/fj.202502066RR

Figure Lengend Snippet: TRIM72 abundance is associated with MuRF1 upregulation in C2C12 myotubes. (A) TRIM72 protein levels are reduced in MuRF1 knockout C2C12 myotubes. Total lysates from WT (wild type) or two different MuRF1 knockout (KO 1 and 2) C2C12 myotubes were analyzed by immunoblotting and probed with the indicated antibodies. Immunoblots were representative of four independent experiments. (B) Quantification of TRIM72 protein abundance in WT and MuRF1 KO C2C12 myotubes. Quantitative data from (A) were normalized to WT and subjected to one‐way analysis of variance (ANOVA) before Dunnett's multiple comparisons post hoc test analysis. Error bars indicate the mean ± standard deviation ( n = 4). **** p < 0.0001 compared to WT. (C) Transient overexpression of MuRF1 restored TRIM72 protein abundance in MuRF1 KO myotubes. Total lysates of C2C12 myotubes from WT or MuRF1 KO with and without transient transfection of 3xFLAG‐MuRF1 for 48 h were analyzed by immunoblotting and probed with the indicated antibodies. Immunoblots were representative of four independent experiments. (D) Quantification of TRIM72 protein abundance in WT and MuRF1 KO with and without transient transfection of 3xFLAG‐MuRF1. Quantitative data from (C) were normalized to WT and subjected to one‐way analysis of variance (ANOVA) before Dunnett's multiple comparisons post hoc test analysis. Error bars indicate the mean ± standard deviation ( n = 4). **** p < 0.0001 compared to WT. (E) Representative blots of two independent experiments showing MuRF1 and TRIM72 protein abundance during differentiation time course for up to 13 days in C2C12 cells. Total lysates from C2C12 cells at the indicated days of differentiation were analyzed by immunoblotting and probed with the indicated antibodies. (F) Bar chart analysis of MuRF1 and TRIM72 protein abundance during differentiation time course in C2C12 cells. Quantitative data from (E) were normalized to day 0 and presented as a bar chart. n = 2 in each time point.

Article Snippet: L6 rat and C2C12 mouse skeletal muscle myoblasts were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knock-Out, Western Blot, Quantitative Proteomics, Standard Deviation, Over Expression, Transfection

Journal: The FASEB Journal

Article Title: MuRF1 Partners With TRIM72 to Impair Insulin Signaling in Skeletal Muscle Cells

doi: 10.1096/fj.202502066RR

Figure Lengend Snippet: Dexamethasone increases TRIM72 and MuRF1 protein abundance while decreasing proximal insulin signaling in C2C12 myotubes. Total lysates from C2C12 myotubes treated with 0.1% ethanol as controls (con) or with indicated concentrations of dexamethasone (Dex) for 24 h were analyzed by immunoblotting and probed with the indicated antibodies. (A) Representative blots showing the effect of dexamethasone on TRIM72, MuRF1 proteins, and proximal insulin signaling in C2C12 myotubes. Immunoblots were representative of four independent experiments. (B, C, E–H) Quantitative data of TRIM72, MuRF1, and IRS1 protein abundances and proximal insulin signaling, including Akt and p70 S6K1 phosphorylation status. Quantitative data were normalized to control (con) and subjected to one‐way analysis of variance (ANOVA) before Dunnett's multiple comparisons post hoc test analysis. Error bars indicate the mean ± standard deviation ( n = 4 independent experiments). Statistical analysis was considered significant at * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with control. (D) Scatter plot comparing the TRIM72 protein abundance and MuRF1 protein abundance in C2C12 myotubes treated with dexamethasone. Each data point represents data from n = 4 independent experiments across 6 treatment groups (total n = 24), with color indicating dexamethasone treatment concentration (μM).

Article Snippet: L6 rat and C2C12 mouse skeletal muscle myoblasts were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Quantitative Proteomics, Western Blot, Phospho-proteomics, Control, Standard Deviation, Concentration Assay

Journal: The FASEB Journal

Article Title: MuRF1 Partners With TRIM72 to Impair Insulin Signaling in Skeletal Muscle Cells

doi: 10.1096/fj.202502066RR

Figure Lengend Snippet: MuRF1 KO prevents dexamethasone‐induced downregulation of proximal insulin signaling and insulin‐stimulated glucose uptake in C2C12. (A) Immunoblots from three independent experiments showing IRS1, p‐Akt (Ser473), MuRF1, and TRIM72 in WT and MuRF1 KO C2C12 myotubes treated with and without dexamethasone. Total lysates from WT or MuRF1 KO C2C12 myotubes treated with and without 1 μM dexamethasone (Dex) for 24 h were analyzed by immunoblotting before probing with the appropriate antibodies. (B–E) Figures showing quantitative data of IRS1 protein abundance (B), Akt Ser473 phosphorylation (C), MuRF1 (D), and TRIM72 (E) protein abundances. Quantitative data from (A) were normalized to wild‐type control (WT con) and subjected to a two‐way ANOVA before Tukey's multiple comparisons post hoc test analysis. Error bars indicate the mean ± standard deviation ( n = 3). * p < 0.05, ** p < 0.01, compared to WT con. nd: Non‐detectable. (F) Insulin‐stimulated 2‐Deoxyglucose (2DG) uptake in WT and MuRF1 KO treated with or without dexamethasone. C2C12 WT and MuRF1 KO myotubes pre‐treated with or without 1 μM dexamethasone for 24 h were serum‐starved overnight, stimulated by 1 μM insulin for 1 h before measuring 2DG uptake for 30 min. Results were presented as glucose uptake (2DG fold‐changes) relative to external controls (without insulin stimulation). Data were analyzed by a two‐way ANOVA before Tukey's multiple comparisons post hoc test analysis. Error bars indicate the mean ± standard deviation ( n = 3 independent experiments). ** p < 0.01 compared with insulin‐stimulated control.

Article Snippet: L6 rat and C2C12 mouse skeletal muscle myoblasts were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Quantitative Proteomics, Phospho-proteomics, Control, Standard Deviation

Journal: The FASEB Journal

Article Title: MuRF1 Partners With TRIM72 to Impair Insulin Signaling in Skeletal Muscle Cells

doi: 10.1096/fj.202502066RR

Figure Lengend Snippet: Restoration of TRIM72 in C2C12 MuRF1 KO myotubes decreases proximal insulin signaling. (A) Immunoblots from four independent experiments showing IRS1, p‐Akt (Ser473), p70 S6K1 (T389), TRIM72, and MuRF1 in WT and MuRF1 KO C2C12 myotubes. Total lysates of C2C12 myotubes from WT or MuRF1 KO with and without transient transfection of FLAG‐TRIM72 for 48 h were analyzed by immunoblotting and probed with the appropriate antibodies. (B–D) Figures showing quantitative data of IRS1 protein abundance (B), Akt Ser (473) phosphorylation (C), and p70 S6K1 (T389) phosphorylation (D). Quantitative data from (A) were normalized to wild‐type (WT) and subjected to a one‐way ANOVA before Dunnett's multiple comparisons post hoc test analysis. Error bars indicating: Mean ± standard deviation ( n = 4 independent experiments). * p < 0.05, ** p < 0.01, compared to Wild Type. ns, Non‐significant.

Article Snippet: L6 rat and C2C12 mouse skeletal muscle myoblasts were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Transfection, Quantitative Proteomics, Phospho-proteomics, Standard Deviation