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