c2c12  (ATCC)

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Establishment and characterization of a fibrotic macrophage-muscle fibrosis model. a) Brightfield image of unstimulated RAW264.7 cells in the top chamber of a RAW-C2C12 coculture system, b) Brightfield image of C2C12 cells in the bottom chamber under basal coculture conditions, c) Fluorescence image of C2C12 cells in the bottom chamber exhibiting myotube morphology, d) Fluorescence image confirming the absence of fibrotic marker expression in C2C12 cells under basal conditions, e) Brightfield image of LPS-activated RAW264.7 cells in the top chamber, f) Brightfield image of C2C12 cells in the bottom chamber displaying a myofibroblast-like morphology post-LPS activation, g) Fluorescence image of phenotypic transition of C2C12 cells to myofibroblast-like cells, h) Fluorescence image demonstrating fibrotic protein expression in LPS-treated C2C12 cells (magenta arrow indicates CD38 cells). Immunostaining images: In panels c) and g), TGF-β, DAPI, and actin are colored red, blue, and green, respectively. In panels d) and h), COL1, α-SMA, CD38, and DAPI are colored red, blue, magenta, and green, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Fluorescence, Marker, Expressing, Activation Assay, Immunostaining

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Study of myogenesis through myotube formation. Brightfield images taken using a 10× objective: a) C2C12 RAW, b) C2C12 RAW LPS, c) C2C12 RAW LPS PFD, d) C2C12 RAW LPS- H, e) C2C12 RAW LPS - PH, indicating myotube formation, f) Graph representing the number of myotubes. The data points are presented as mean ± SEM (n = 4). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. The scale bar denotes 50 μm # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Immunofluorescence images showing the expression of myogenesis (MyHC) or fibrogenesis (α-SMA and COL) markers. a) C2C12, b) C2C12 RAW, c) C2C12 RAW LPS, d) C2C12 RAW LPS PFD, e) C2C12 RAW LPS - H, f) C2C12 RAW LPS - PH. Semiquantitative expression analysis of the myogenesis/fibrosis markers. g) MyHC/α-SMA and h) MyHC/α-SMA. I) MyHC/α-SMA/DAPI staining, II) MyHC/COL1/DAPI staining. The data points are presented as mean ± SEM (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. The scale bar denotes 50 μm # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Immunofluorescence, Expressing, Staining, Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Gene analysis of fibrogenesis and myogenesis markers. a) COL1, b) MMP8, c) Acta2, d) LOX, and e) MyoG using qPCR. The data points are presented as mean ± SEM (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: AFM images of cells in contact mode in PBS after treatment. a) C2C12 RAW, b) C2C12 RAW LPS, c) C2C12 RAW LPS PFD, d) C2C12 RAW LPS - H, e) C2C12 RAW LPS - PH, (f) graph representing nanoindentation measurements denoting the stiffness of the cell. The data points are presented as mean ± SEM (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Mitochondrial metabolism study using a Seahorse extracellular analyzer. Mitochondrial stress test, a) Scheme representing the mitochondrial stress test profile, b) OCR graphs of the inflammation model, c) OCR graphs of the treatment conditions, d) Proton leak measurements, e) ATP production linked to O 2 consumption, f) Maximal respiration, and g) Spare capacity. The data points are presented as mean ± SEM. (n = 3). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # indicates significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively; & and NS indicate significant and non-significant differences between free drug (PFD) and the sustained delivery system (C2C12 RAW LPS-PH), respectively.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: Transmission electron micrographs depicting cell morphology and mitochondrial morphology. (a) C2C12 RAW co-culture with a black box indicating the native muscle cell structure. (b) C2C12 RAW LPS, with a black box highlighting C2C12 phenotypically transitioned to myofibroblast-like cell structures. TEM images of mitochondria within single cells are shown for (c) C2C12 RAW, (d) C2C12 RAW LPS, and (e) C2C12 RAW LPS - PH (PH - Pirfenidone loaded hydrogel) treatment. Yellow arrows point to mitochondria. (The data points are presented as mean ± SEM. (n = 4). A two-tailed Student's t-test was performed to assess the statistical significance of differences between the control and treatment groups. # and ns indicates significant and non-significant differences between control (C2C12 RAW) and fibrotic (C2C12 RAW LPS) groups; ∗ and ns indicate the significance and non-significance between the fibrotic group and treatment groups (PFD or hydrogels), respectively. Scale bars in figures (a, b) represent 5 μm, c) 1 μm, d, e) 2 μm, and the insets are of a 500 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Transmission Assay, Co-Culture Assay, Two Tailed Test, Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: a) Comparative transcriptomic profiles of the control (C2C12 RAW), fibrotic (C2C12 RAW LPS), and treatment (C2C12 RAW LPS - PH) groups, b) Volcano plot of DEGs in the fibrotic (C2C12 RAW LPS) vs control (C2C12 RAW) groups, and c) Volcano plot of DEGs in the treated (C2C12 RAW LPS - PH) vs fibrotic (C2C12 RAW LPS) groups.

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Control

Journal: Materials Today Bio

Article Title: Injectable antifibrotic drug-loaded hydrogels reduce fibrosis and restore myogenesis by enhancing mitochondrial metabolism and cell mechanics in an in vitro coculture model

doi: 10.1016/j.mtbio.2026.103033

Figure Lengend Snippet: A comparison analysis depicting the a) biological functions and b) canonical pathways associated with fibrosis and healthy muscle regeneration in the control (C2C12 RAW) vs. fibrotic (C2C12 RAW LPS) vs. treatment (C2C12 RAW LPS - PH) groups. The intensity of the heatmap color corresponds to the activation z-score due to the involvement of upregulated and downregulated genes. The supplementary table listing the pathways along with their corresponding z-score values is included in the supplementary section (Table ST1).

Article Snippet: C2C12 cells and RAW 264.7 cells were acquired from the American Type Culture Collection (ATCC), USA.

Techniques: Comparison, Control, Activation Assay

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: Comparative analysis of conventional and brush-assisted bioprinting on cellular behavior. (a) Schematic illustration of shear stress distribution in normal versus brush-assisted printing. (b) Overview of the brush-assisted printing setup. (c) SEM images of collagen fibrils and fluorescence images showing cell viability (live/dead) and cytoskeletal organization (DAPI/phalloidin) of C2C12 myoblasts. Quantification of (d) cell viability post-printing (n = 4), (e) cell metabolic activity (MTT assay, in situ /day 3/day 7, n = 4), (f) nuclei aspect ratio (n = 180), (g) orientation factor (n = 3), and (h) F-actin–positive area at day 3 (n = 10). (i) Comparing normal and brush-assisted printing the mechanotransduction pathways activated by shear stress and collagen alignment. (j) Heatmap of relative gene expression ( YAP, TAZ, AKT1, PIEZO1, PI3K, and CAPN2 ) after 7 days of culture (n = 4). (k) Agarose gel electrophoresis of PCR products from cells cultured on normal versus brush-printed scaffolds for 7 days. (l) Schematic illustration of blocking mechano-sensing ion channel with GsMTx-4. (m) Relative gene expression levels associated with mechanosensing channel and ca 2+ pathway (n = 5), (n) Hippo pathway (n = 5), (o) PI3K-AKT pathway (n = 5). Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). Abbreviation: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (cell metabolic activity assay); F-actin, Filamentous actin; PI3K , Phosphoinositide 3-kinase; CAPN2 , Calcium-activated neutral protease 2.

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: Shear, Fluorescence, Activity Assay, MTT Assay, In Situ, Gene Expression, Agarose Gel Electrophoresis, Cell Culture, Blocking Assay, Standard Deviation, Metabolic Assay

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: Physical and biological evaluation of bioconstructs reinforced with straight (CSP) and coiled (CCP) PCL fibers. (a) Schematic illustration and optical images of flow tests with Col (collagen-only), CSP, and CCP bioinks, in which droplets were tilted at 90° for 10 min. (b) Quantification of droplet outflow (n = 10). Rheological measurements of bioinks: (c) storage modulus (G′) from frequency sweep (n = 3), (d) G′ and G″ from temperature sweep (n = 3), and (e) G′ and G″ under cyclic stress loading (10 and 200 Pa) showing viscoelastic recovery (n = 3). (f) SEM images of CCP constructs highlighting coiled PCL fiber and aligned collagen fibrils. (g) Optical image, live/dead staining, DAPI/phalloidin staining, and orientation factor analysis of C2C12 cells and PCL microfibers. Quantification of (h) cell viability (live/dead, n = 4), (i) F-actin–positive area (n = 20), and (j) metabolic activity (MTT assay, in situ /day 3/day 7, n = 4). Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗).

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: Construct, Staining, Activity Assay, MTT Assay, In Situ, Standard Deviation

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: In vitro myogenic differentiation of C2C12 cells cultured on Col, CSP and CCP scaffolds. (a) Live/dead staining at in situ , DAPI/phalloidin staining at day 3, and DAPI/ MHC staining at day 14. (b) Quantification of cell viability from live/dead assays (n = 4). (c) Nuclei orientation factor after 7 days of culture (n = 4). (d) Nuclei aspect ratio (n = 20). (e) F-actin positive area (n = 4). (f) Quantification of MHC fusion index (left, n = 5) and MHC maturation rate (right, n = 5). (g) Relative gene expression analysis and (h) agarose gel electrophoresis regarding mechanotransduction-related genes ( CAPN2, PIEZO1, RhoA, YAP, and TAZ ) (n = 4). (i) Western blot analysis of PIEZO1 . (j) Schematic illustrating differentiation progression and major genes involved at each stage. (k) Heatmap and (l) agarose gel electrophoresis of PCR products showing relative expression of myogenic markers ( MYF5, MYOD1, MYOG, MHC, MYH2, and MYH4 ) after 21 days of culture (n = 4). (m) Western blot analysis of MHC . Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). Abbreviation: MYF5 , Myogenic factor 5; MYOD1 , Myogenic differentiation 1; MYOG , Myogenin; MHC , Myosin heavy chain; MYH2 , Myosin heavy chain 2; MYH4 , Myosin heavy chain 4.

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: In Vitro, Cell Characterization, Cell Culture, Staining, In Situ, Gene Expression, Agarose Gel Electrophoresis, Western Blot, Expressing, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Veronicastrum sibiricum (L.) Pennell extract alleviates inflammation-induced muscle atrophy through NLRP3 inflammasome regulation and mitochondrial function restoration

doi: 10.3892/mmr.2026.13857

Figure Lengend Snippet: Effects of VSE on inflammation-induced muscle atrophy in C2C12 myotubes. All experiments were carried out after C2C12 myoblasts had differentiated into myotubes for 4 days. C2C12 myotubes were pretreated with 0 (LPS only), 1.25, 2.5 or 5 µg/ml VSE for 3 h and then co-treated with 500 ng/ml LPS for (B, D and E) 24 h and (C) 48 h. Controls received no treatments. (A) C2C12 myotubes were exposed to 0–10 µg/ml VSE for 48 h to assess cytotoxicity. (B) mRNA levels of muscle-specific E3 ubiquitin ligases MuRF1 and atrogin-1 were measured using reverse transcription-quantitative PCR. (C) Giemsa-stained images of C2C12 myotubes showing changes in relative fiber width after treatment. CON, untreated control; LPS, 500 ng/ml; 1.25, 2.5 and 5, co-treatment with LPS (500 ng/ml) and VSE at the indicated concentrations (µg/ml). Analysis was performed using ImageJ (National Institutes of Health). Scale bar, 100 µm. Western blot analysis of (D) MuRF1, MaFbx and MyHC protein levels, and (E) phosphorylated Akt and Foxo3a, including membrane images and semi-quantitative analysis using ImageJ. All data are presented as the mean ± SD of triplicate results and statistical analysis was carried out using one-way ANOVA with Tukey's post hoc test. ## P<0.01 and #### P<0.0001 vs. control group; *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001 vs. LPS-treated group. MuRF1, muscle-specific RING finger protein 1; MaFbx, muscle atrophy F-box protein; MyHC, myosin heavy chain; CON, control; LPS, lipopolysaccharide; VSE, Veronicastrum sibiricum seed extract; p-, phosphorylated; ns, not significant.

Article Snippet: Murine C2C12 myoblast cells were obtained from American Type Culture Collection.

Techniques: Ubiquitin Proteomics, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining, Control, Western Blot, Membrane

Journal: Molecular Medicine Reports

Article Title: Veronicastrum sibiricum (L.) Pennell extract alleviates inflammation-induced muscle atrophy through NLRP3 inflammasome regulation and mitochondrial function restoration

doi: 10.3892/mmr.2026.13857

Figure Lengend Snippet: VSE downregulates the NLRP3 inflammasome. C2C12 myotubes were pretreated with VSE at 0 (LPS only), 1.25, 2.5 and 5 µg/ml for 3 h and then co-treated with 500 ng/ml LPS for 24 h. Controls received no treatments. (A) mRNA expression levels of NLRP3 , the NLRP3 inflammasome initiator, were analyzed using reverse transcription-quantitative PCR. (B) Protein expression levels of factors associated with the NLPR3 pathway, a major pyroptosis pathway, were analyzed using western blotting and ImageJ. All data are presented as the mean ± SD of triplicate results and statistical analysis was carried out using one-way ANOVA with Tukey's post hoc test. #### P<0.0001, ### P<0.001, ## P<0.01 vs. control; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. LPS treatment. NLRP3, NLR family pyrin domain containing 3; GSDMD, gasdermin-D; LPS, lipopolysaccharide; VSE, Veronicastrum sibiricum seed extract.

Article Snippet: Murine C2C12 myoblast cells were obtained from American Type Culture Collection.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Veronicastrum sibiricum (L.) Pennell extract alleviates inflammation-induced muscle atrophy through NLRP3 inflammasome regulation and mitochondrial function restoration

doi: 10.3892/mmr.2026.13857

Figure Lengend Snippet: VSE reduces the secretion of proinflammatory cytokines. C2C12 myotubes were pretreated with VSE at 0 (LPS only), 1.25, 2.5 and 5 µg/ml for 3 h and then co-treated with 500 ng/ml LPS for 24 h. Controls received no treatments. (A) mRNA expression levels of the proinflammatory cytokines TNF-α and IL-1β were analyzed using reverse transcription-quantitative PCR. (B) Protein expression levels of the proinflammatory cytokines TNF-α and IL-1β in the supernatant of C2C12 myotube cultures were analyzed using ELISA. All data are presented as the mean ± SD of triplicate results and statistical analysis was carried out using one-way ANOVA with Tukey's post hoc test. #### P<0.0001 vs. control; ***P<0.001, ****P<0.0001 vs. LPS treatment. A450, absorbance at 450 nm; LPS, lipopolysaccharide; VSE, Veronicastrum sibiricum seed extract.

Article Snippet: Murine C2C12 myoblast cells were obtained from American Type Culture Collection.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Molecular Medicine Reports

Article Title: Veronicastrum sibiricum (L.) Pennell extract alleviates inflammation-induced muscle atrophy through NLRP3 inflammasome regulation and mitochondrial function restoration

doi: 10.3892/mmr.2026.13857

Figure Lengend Snippet: VSE exerts antioxidant effects in C2C12 myotubes. C2C12 myotubes were pretreated with 0 (LPS only), 1.25, 2.5 and 5 µg/ml VSE for 3 h and then co-treated with 500 ng/ml LPS for 24 h. (A) Protein expression levels of antioxidant-related factors HO-1, Nrf2 and Keap1, and mitochondrial metabolism-related factor PGC-1α were analyzed using western blotting. (B) Images (left) were obtained under a fluorescence microscope to investigate the generation of mitochondrial ROS using ImageJ (right). Scale bar, 100 µm. All data are presented as the mean ± SD of triplicate results, and statistical analysis was carried out using one-way ANOVA with Tukey's post hoc test. # P<0.05, ## P<0.01, #### P<0.0001 vs. control; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. LPS treatment. LPS, lipopolysaccharide; VSE, Veronicastrum sibiricum seed extract; ROS, reactive oxygen species; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2; Keap1, kelch like ECH associated protein 1; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1α; CON, control.

Article Snippet: Murine C2C12 myoblast cells were obtained from American Type Culture Collection.

Techniques: Expressing, Western Blot, Fluorescence, Microscopy, Control

Journal: Molecular Medicine Reports

Article Title: Veronicastrum sibiricum (L.) Pennell extract alleviates inflammation-induced muscle atrophy through NLRP3 inflammasome regulation and mitochondrial function restoration

doi: 10.3892/mmr.2026.13857

Figure Lengend Snippet: VSE exerts protective effects on mitochondrial function, reducing functional impairment under inflammatory conditions. C2C12 myotubes were pretreated with 0 (LPS only), 1.25, 2.5 and 5 µg/ml VSE for 3 h and then co-treated with 500 ng/ml LPS for 24 h. (A) Protein expression levels of the mitochondrial dynamics-related OXPHOS genes, including CI-CV, were analyzed using western blotting. (B) JC-1 staining was carried out to assess the mitochondrial membrane potential. Representative images of JC-1 fluorescence (red/green) were analyzed using ImageJ (National Institutes of Health). Scale bar, 25 µm. All data are presented as the mean ± SD of triplicate results and statistical analysis was carried out using one-way ANOVA with Tukey's post hoc test. ## P<0.01, #### P<0.0001 vs. control; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. LPS treatment. LPS, lipopolysaccharide; VSE, Veronicastrum sibiricum seed extract; CI-CV, complexes I–V; OXPHOS, oxidative phosphorylation system; CON, control.

Article Snippet: Murine C2C12 myoblast cells were obtained from American Type Culture Collection.

Techniques: Functional Assay, Expressing, Western Blot, Staining, Membrane, Fluorescence, Control, Phospho-proteomics

Journal: Journal of Advanced Research

Article Title: Exercise improves hypobaric hypoxia-induced skeletal muscle dysfunction via Sirt1-Mediated myotube and mitochondrial remodeling

doi: 10.1016/j.jare.2025.08.022

Figure Lengend Snippet: Hypobaric hypoxia-induces skeletal muscle dysfunction: characterized by decreased muscle mass and impaired physical performance. (A) The schematic of the experimental setup for the 45-day chronic hypobaric hypoxia murine model. (B) Following 45-day hypobaric hypoxia exposure, mice exhibited reduced four-limb grip strength compared to normoxia controls. n = 5–7/group. (C) Bars represent mean running time on a motorized treadmill. n = 8/group. (D) Bars represent mean rotarod test time on a rotarod latency. n = 10/group. (E) Measurements of muscle tetanic contraction show reduced force in the hypobaric hypoxia group. n = 13–15/group. (F) Representative TA muscles from indicated mice. TA muscle weight was normalized to body mass after hypobaric hypoxia exposure. n = 7/group. (G) Representative haematoxylin and eosin (H&E) staining of TA muscle sections and quantification of CSA. Scale bar: 50 μm. n = 3/group. (H) Representative images of MyHC immunofluorescence of C2C12 cells under control and hypoxia. Measurements of myotube diameter. Scale bar: 50 μm. n = 100 myotubes per condition. (I) Immunoblot analysis and quantification of protein expression in hypoxic C2C12 cells using the indicated antibodies. n = 6/group. * p < 0.05; * *p < 0.01; *** p < 0.001. Unpaired two-tailed t -test compared two groups. Two-way ANOVA was employed for comparisons involving experimental factors with more than two levels.

Article Snippet: C2C12 myoblasts (American Type Culture Collection, CRL-1772) were cultured in DMEM supplemented with 10 % FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL).

Techniques: Muscles, Staining, Immunofluorescence, Control, Western Blot, Expressing, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: Exercise improves hypobaric hypoxia-induced skeletal muscle dysfunction via Sirt1-Mediated myotube and mitochondrial remodeling

doi: 10.1016/j.jare.2025.08.022

Figure Lengend Snippet: Chronic hypobaric hypoxia exposure impairs the mitochondrial function of skeletal muscle. (A) GO analysis of TA muscle proteomes from normoxia and hypobaric hypoxia groups, profiled by LC-MS/MS. n = 6/group. (B) Heatmap analysis of mitochondrial-related proteins identified by proteomics. (C) Immunoblot analysis and quantification of OXPHOS complexes in TA muscle lysates using the Total OXPHOS Rodent Antibody Cocktail. n = 6/group. (D) Mitochondrial DNA content in TA muscle assessed by qPCR (primers: mitochondria-encoded mtDNA vs. Nuclear-encoded Rbm15). n = 3/group. (E) Mitochondrial membrane potential in C2C12 cells under control and hypoxia using MitoTracker® Deep Red FM. Quantification of the average optical density of MitoTracker. Scale bar: 50 μm/100 μm. (F) Immunoblot analysis and quantification of OXPHOS complexes in C2C12 cells. n = 4/group. * *p < 0.01; *** p < 0.001. Unpaired two-tailed t -test compared two groups. Two-way ANOVA was employed for comparisons involving experimental factors with more than two levels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: C2C12 myoblasts (American Type Culture Collection, CRL-1772) were cultured in DMEM supplemented with 10 % FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Membrane, Control, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: Exercise improves hypobaric hypoxia-induced skeletal muscle dysfunction via Sirt1-Mediated myotube and mitochondrial remodeling

doi: 10.1016/j.jare.2025.08.022

Figure Lengend Snippet: E3 ubiquitin ligase facilitates degradation of the NAD + -dependent deacetylase Sirt1 under hypoxic conditions. (A) The GO enrichment and pathway analysis of TA muscle proteomes. (B) Immunoblot analysis and quantification of muscle-inhibitory factor expression in TA muscle. n = 6/group. (C) The atrogenes expression level in TA muscle assessed by qPCR. n = 3/group. (D) Immunoblot analysis and quantification of Trim63 in C2C12 cells. n = 6/group. (E) The atrogenes expression level in C2C12 cells assessed by qPCR. n = 3/group. (F) Immunoblot analysis and quantification of Sirt1 expression in TA muscle and C2C12 cells. n = 6/group. (G) Immunoprecipitation analysis of Sirt1 ubiquitination upon co-transfection with Ub-K48 plasmids in C2C12 cells. After 48 h hypoxia exposure, cells were treated with 10 μM MG132 for 24 h. (H) Immunoblot analysis of the expression of Nedd4 and Mdm2 in the whole cell lysates (WCL) of C2C12 cells. (I) Immunoprecipitation analysis of the interaction between E3 ubiquitin ligases and Sirt1 after hypoxic stimulation. * *p < 0.01; *** p < 0.001. Unpaired two-tailed t -test compared two groups. Two-way ANOVA was employed for comparisons involving experimental factors with more than two levels.

Article Snippet: C2C12 myoblasts (American Type Culture Collection, CRL-1772) were cultured in DMEM supplemented with 10 % FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL).

Techniques: Ubiquitin Proteomics, Histone Deacetylase Assay, Western Blot, Expressing, Immunoprecipitation, Cotransfection, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: Exercise improves hypobaric hypoxia-induced skeletal muscle dysfunction via Sirt1-Mediated myotube and mitochondrial remodeling

doi: 10.1016/j.jare.2025.08.022

Figure Lengend Snippet: Overexpression of Sirt1 attenuates hypoxia-induced myotube atrophy and mitochondrial dysfunction. (A) Immunoblot analysis and quantification of Sirt1 in C2C12 cells transfected with pLV3-CMV-Sirt1 (OE-Sirt1) or empty vector. Myotubes were cultured under control or hypoxia conditions for 72h. n = 5/group. (B) Representative images of MyHC immunofluorescence in C2C12 cells infected with pLV3-CMV-Sirt1 or empty vector under control or hypoxia conditions. Scale bar: 100 μm. Measurements of myotube diameter. n = 100 myotubes per condition. (C) Immunoblot analysis and quantification of Trim63 and Myogenin in C2C12 cells transfected with the indicated constructs. n = 5–6/group. (D) Representative images of mitochondrial membrane potential in myotubes transfected with the indicated constructs using JC-10. Scale bar: 100 μm. Quantification of the ratio of JC-10 aggregates to monomers, i.e., the ratio of red to green fluorescence. n = 3/group. (E) Representative images and quantification of ROS levels in myotubes transfected with the indicated constructs using the DHE probe. Scale bar: 100 μm. n = 3/group. (F) Mitochondrial DNA content and ATP production in C2C12 cells transfected with the indicated constructs. n = 3/group. (G) Graphical representation of oxygen consumption rates (OCR) and ATP production in treated cells is shown. n = 3/group. (H) Immunoblot analysis and quantification of mitochondrial-related protein in C2C12 cells transfected with the indicated constructs. n = 4/group. (I) Immunoblot analysis and quantification of PGC-1α expression in TA muscle and C2C12 cells. n = 5–6/group. (J) Immunoblot analysis and quantification of PGC-1α in C2C12 cells transfected with the indicated constructs. n = 5/group. (K) Immunoprecipitation using PGC-1α antibody and immunoblot analysis of the indicated proteins. * p < 0.05; * *p < 0.01; *** p < 0.001. Unpaired two-tailed t -test compared two groups. One-way ANOVA was used for comparisons involving more than two groups. Two-way ANOVA was employed for comparisons involving experimental factors with more than two levels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: C2C12 myoblasts (American Type Culture Collection, CRL-1772) were cultured in DMEM supplemented with 10 % FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL).

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Cell Culture, Control, Immunofluorescence, Infection, Construct, Membrane, Fluorescence, Expressing, Immunoprecipitation, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: Exercise improves hypobaric hypoxia-induced skeletal muscle dysfunction via Sirt1-Mediated myotube and mitochondrial remodeling

doi: 10.1016/j.jare.2025.08.022

Figure Lengend Snippet: Exercise-mimetic stimulation activates myotube function improvement via Sirt1-PGC-1α/FoxO3a in C2C12 cells. (A) C2C12 myotubes were treated with the AMPK activator AICAR (0.25 mM) for 12 h, then exposed to hypoxia for 72 h. Immunoblot analysis and quantification of Sirt1 and p-AMPK in C2C12 cells were performed. n = 6/group. (B) Representative images of MyHC immunofluorescence in C2C12 cells treated with AICAR under control or hypoxia conditions and measurements of myotube diameter. Scale bar: 100 μm. n = 100 myotubes per condition. (C) Immunoblot analysis and quantification of Trim63 in C2C12 cells treated with AICAR under control or hypoxia conditions. n = 6/group. (D) Representative images of mitochondrial membrane potential in myotubes treated with AICAR under control or hypoxia using JC-10. Scale bar: 100 μm. Quantification of the ratio of JC-10 aggregates to monomers. n = 3/group. (E) Representative images of ROS levels and quantification of the average optical density of DCFH-DA in myotubes treated with AICAR. Scale bar: 100 μm. n = 3/group. (F) Mitochondrial DNA content (n = 6/group) and ATP production in C2C12 cells treated with AICAR(n = 3/group). (G) The redox status of myotubes treated with AICAR was characterized by the ratio of NAD + /NADH. n = 3/group. (H) Graphical representation of OCR and ATP production in treated cells is shown. n = 3/group. (I) Immunoblot analysis and quantification of PGC-1α in C2C12 cells treated with AICAR. n = 6/group. (J) Immunoprecipitation using PGC-1α antibody and immunoblot analysis of the indicated proteins were performed. (K) Immunoblot analysis and quantification of FoxO3a in C2C12 cells treated with AICAR. n = 6/group. (L) Immunoprecipitation using FoxO3a antibody and immunoblot analysis of the indicated proteins were performed. * p < 0.05; * *p < 0.01; *** p < 0.001. Unpaired two-tailed t -test compared two groups. One-way ANOVA was used for comparisons involving more than two groups. Two-way ANOVA was employed for comparisons involving experimental factors with more than two levels.

Article Snippet: C2C12 myoblasts (American Type Culture Collection, CRL-1772) were cultured in DMEM supplemented with 10 % FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL).

Techniques: Western Blot, Immunofluorescence, Control, Membrane, Immunoprecipitation, Two Tailed Test

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Elevated Circulating Ceramides 18:0 and 24:1 as a Risk Factor for Sarcopenia: In Vitro, Animal, and Clinical Evidence

doi: 10.1002/jcsm.70310

Figure Lengend Snippet: C18:0 ceramide impairs in vitro myogenic differentiation. (a) Mouse C2C12 myoblasts were differentiated into myotubes using 2% horse serum in the presence of the indicated concentrations of C18:0 ceramide for 3 days. Myotubes were immunostained with an anti‐MyHC antibody, and nuclei were counterstained with DAPI. Quantitative analyses per field are shown ( n = 4). (b) Western blot and (c) quantitative reverse transcription polymerase chain reaction analyses were performed to assess the expression levels of myogenic markers myogenin and MyHC following 3‐day treatment with 0.1 μM C18:0 ceramide during differentiation ( n = 3). (d) Myoblast migration was evaluated using a Boyden chamber assay, and (e) cell viability was measured with a CCK‐8 assay after treatment with the indicated concentrations of C18:0 ceramide for 6 and 24 h, respectively ( n = 5). (f) The inhibitory effects of C18:0 ceramide on myogenic differentiation were similarly confirmed using primary myoblasts under the same experimental conditions as described in (a). Scale bars: 100 μm (a), 50 μm (d), 100 μm (f). MyHC, myosin heavy chain; DAPI, 4′,6‐diamidino‐2‐phenylindole; OD, optical density. * p < 0.05 vs. untreated control.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, Virginia, United States) were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS), 20 mM HEPES, 2 mM L‐glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all from Life Technologies, Carlsbad, California, United States).

Techniques: In Vitro, Cell Characterization, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Expressing, Migration, Boyden Chamber Assay, CCK-8 Assay, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Elevated Circulating Ceramides 18:0 and 24:1 as a Risk Factor for Sarcopenia: In Vitro, Animal, and Clinical Evidence

doi: 10.1002/jcsm.70310

Figure Lengend Snippet: The inhibitory effects of C18:0 ceramide on myogenesis are mediated by increased intracellular ROS generation. (a) Mouse C2C12 myoblasts were differentiated into myotubes using 2% horse serum in the presence of the indicated concentrations of C18:0 ceramide for 3 days. Intracellular ROS levels were assessed using the fluorescent probe chloromethyl‐2′,7′‐dichlorofluorescein diacetate (CM‐H 2 DCFDA) ( n = 5). (b,c) C2C12 cells were differentiated into myotubes with 2% horse serum in the presence or absence of 0.1 μM C18:0 ceramide and/or 1 mM NAC for 3 days. (b) Intracellular ROS levels were measured using H 2 DCFDA ( n = 3). (c) Myotubes were stained with anti‐MyHC antibody, and nuclei were counterstained with DAPI. Quantitative analyses of myogenic parameters per field are shown ( n = 3). (d) Expression level of MyHC was assessed by western blotting under the same conditions ( n = 3). (e,f) C2C12 cells were treated with 0.1 μM C18:0 ceramide and/or 1 mM NAC for 1 day ( n = 3). (e) SA‐β‐gal‐positive cells (%) per field were quantified, and representative images of SA‐β‐gal staining are shown. SA‐β‐gal‐positive cells appear blue. (f) Western blot analyses were performed to assess p21 and p16 expression levels. β‐Tubulin served as a loading control. Scale bars: 100 μm (b,c), 20 μm (e). ROS, reactive oxygen species; NAC, N‐acetyl cysteine; MyHC, myosin heavy chain; DAPI, 4′,6‐diamidino‐2‐phenylindole; SA‐β‐gal, senescence‐associated β‐galactosidase. * p < 0.05 vs. untreated control; # p < 0.05 vs. 0.1 μM C18:0 ceramide.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, Virginia, United States) were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS), 20 mM HEPES, 2 mM L‐glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all from Life Technologies, Carlsbad, California, United States).

Techniques: Staining, Expressing, Western Blot, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Elevated Circulating Ceramides 18:0 and 24:1 as a Risk Factor for Sarcopenia: In Vitro, Animal, and Clinical Evidence

doi: 10.1002/jcsm.70310

Figure Lengend Snippet: C24:1 ceramide impairs in vitro myogenic differentiation. (a) Mouse C2C12 myoblasts were differentiated into myotubes using 2% horse serum in the presence of the indicated concentrations of C24:1 ceramide for 3 days. Myotubes were immunostained with an anti‐MyHC antibody, and nuclei were counterstained with DAPI. Quantitative analyses per field are shown ( n = 4). (b) Western blot and (c) quantitative reverse transcription polymerase chain reaction analyses were conducted to evaluate the expression of myogenic markers, myogenin and MyHC, following 3‐day treatment with 0.1 μM C24:1 ceramide during differentiation ( n = 3). (d) Myoblast migration was assessed using a Boyden chamber assay, and (e) cell viability was measured using a CCK‐8 assay after treatment with the indicated concentrations of C24:1 ceramide for 6 and 24 h, respectively ( n = 5). (f) The inhibitory effects of C24:1 ceramide on myogenic differentiation were also confirmed in primary myoblasts under the same experimental conditions as described in (a). Scale bars: 100 μm (a), 50 μm (d), 100 μm (f). MyHC, myosin heavy chain; DAPI, 4′,6‐diamidino‐2‐phenylindole; OD, optical density. * p < 0.05 vs. untreated control.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, Virginia, United States) were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS), 20 mM HEPES, 2 mM L‐glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all from Life Technologies, Carlsbad, California, United States).

Techniques: In Vitro, Cell Characterization, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Expressing, Migration, Boyden Chamber Assay, CCK-8 Assay, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Elevated Circulating Ceramides 18:0 and 24:1 as a Risk Factor for Sarcopenia: In Vitro, Animal, and Clinical Evidence

doi: 10.1002/jcsm.70310

Figure Lengend Snippet: The inhibitory effects of C24:1 ceramide on myogenesis are mediated by increased intracellular ROS generation. (a) Mouse C2C12 myoblasts were differentiated into myotubes using 2% horse serum in the presence of the indicated concentrations of C24:1 ceramide for 3 days. Intracellular ROS levels were assessed using the fluorescent probe chloromethyl‐2′,7′‐dichlorofluorescein diacetate (CM‐H 2 DCFDA) ( n = 5). (b,c) C2C12 cells were differentiated into myotubes with 2% horse serum in the presence or absence of 0.1 μM C24:1 ceramide and/or 1 mM NAC for 3 days. (b) Intracellular ROS levels were measured using H 2 DCFDA ( n = 3). (c) Myotubes were stained with anti‐MyHC antibody, and nuclei were counterstained with DAPI. Quantitative analyses of myogenic parameters per field are shown ( n = 3). (d) Expression level of MyHC was assessed by western blotting under the same conditions ( n = 3). (e,f) C2C12 cells were treated with 0.1 μM C24:1 ceramide and/or 1 mM NAC for 1 day ( n = 3). (e) SA‐β‐gal‐positive cells (%) per field were quantified, and representative images of SA‐β‐gal staining are shown. SA‐β‐gal‐positive cells appear blue. (f) Western blot analyses were performed to assess p21 and p16 expression levels. β‐Tubulin served as a loading control. Scale bars: 100 μm (b and c), 20 μm (e). ROS, reactive oxygen species; NAC, N‐acetyl cysteine; MyHC, myosin heavy chain; DAPI, 4′,6‐diamidino‐2‐phenylindole; SA‐β‐gal, senescence‐associated β‐galactosidase. * p < 0.05 vs. untreated control; # p < 0.05 vs. 0.1 μM C24:1 ceramide.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, Virginia, United States) were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS), 20 mM HEPES, 2 mM L‐glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all from Life Technologies, Carlsbad, California, United States).

Techniques: Staining, Expressing, Western Blot, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Elevated Circulating Ceramides 18:0 and 24:1 as a Risk Factor for Sarcopenia: In Vitro, Animal, and Clinical Evidence

doi: 10.1002/jcsm.70310

Figure Lengend Snippet: Ceramide‐induced oxidative stress suppresses ITGB1 signalling and promotes skeletal muscle atrophy in vitro. (a,b) Differentiated C2C12 myotubes were treated with vehicle (Veh), C18:0 ceramide or C24:1 ceramide in the presence or absence of NAC. (a) Western blot analyses were performed to assess ITGB1 protein expression ( n = 3). (b) Myotubes were immunostained with an anti‐ITGB1 antibody, and nuclei were counterstained with DAPI ( n = 5). (c) Western blot analyses were conducted to evaluate the activation status of ITGB1‐associated signalling pathways in myotubes treated with C18:0 or C24:1 ceramide in the presence or absence of the ITGB1‐activating antibody TS2/16. (d) Myotubes were immunostained with an anti‐FoxO1 antibody to assess nuclear localisation following treatment with C18:0 or C24:1 ceramide with or without TS2/16. Nuclei were counterstained with DAPI ( n = 5). (e) mRNA expression levels of MuRF1 and Atrogin‐1 were evaluated by quantitative reverse transcription polymerase chain reaction ( n = 3). (f) Myotube width, myotube area per myotube and myotube size distribution were quantified to assess myotube atrophy following ceramide treatment with or without TS2/16 ( n = 5). Scale bars: 100 μm (b,d). An asterisk (*) indicates a statistically significant difference among groups (b–f).

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, Virginia, United States) were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS), 20 mM HEPES, 2 mM L‐glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all from Life Technologies, Carlsbad, California, United States).

Techniques: In Vitro, Western Blot, Expressing, Activation Assay, Reverse Transcription, Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: The C16orf87 protein is a subunit of the MIER corepressor complex controlling embryonic development and cell migration

doi: 10.1038/s41598-026-50740-7

Figure Lengend Snippet: Knockdown of C16orf87 causes minor changes in the host cell protein profile. ( A ) Alignment of human ( Homo sapiens , UniProtKB accession number Q6PH81 ), mouse ( Mus musculus , UniProtKB accession number Q9CR55 ), and zebrafish ( Danio rerio , UniProtKB accession number Q6DGQ4 ) C16orf87 amino acid sequences. Alignment mismatches are indicated in gray boxes. The underlined sequence represents a possible minimal Akt/PKB kinase consensus recognition motif. A Ser91(S91) phosphorylation site is marked with an asterisk. ( B ) Per-residue confidence (pLDDT) coloring of the top-ranked predicted model of C16orf87. In the inset, the predicted zinc-ribbon domain is shown with the zinc-interacting cysteines (Cys16, Cys19, Cys30, and Cys32) indicated around the zinc ion (Zn 2+ ). The position of the phosphorylated serine (Ser91), a putative alpha-helix between amino acid residues Ser-107 and Ala-126, and the confidently predicted C-terminal alpha-helix between amino acid residues Asp-130 and Ile-153 are also highlighted. The ipTM and pTM values are annotated. N, N-terminus; C, C-terminus. Figure was rendered using ChimeraX (version 1.8, https://www.rbvi.ucsf.edu/chimerax ) ( C ) Western blot (WB) analysis of C16orf87 siRNA (siC16) knockdown in Panc-01, MiaPaCa-2, and C2C12 cell lines. A non-specific, scrambled siRNA (siScr) was used as a control; the WB membrane was probed with the antibodies against C16orf87 and actin. MS-based proteomics analysis of siRNA-treated C2C12 ( D ), MiaPaCa-2 ( E ), and Panc-01 ( F ) cells. Data points corresponding to histones are colored in pink, and statistically significant ( P < 0.05, fold-change > 1) proteins are colored in yellow (mouse cell line C2C12) and green (human cell lines, Panc-01 and MiaPaCa-2).

Article Snippet: Human pancreatic cancer cell lines Panc-01 (ATCC, CRL-1469) and MiaPaCa-2 (ATCC, CRL-1420), mouse skeletal muscle cell line C2C12 (ATCC, CRL-1772), and human cervical cancer cell line HeLa S3 (ATCC, CCL-2.2) were used in this study.

Techniques: Knockdown, Sequencing, Phospho-proteomics, Residue, Western Blot, Control, Membrane

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Deubiquitinase YOD1 Inhibition Suppresses DEX‐ and Denervation‐Induced Muscle Atrophy Through MAFbx Destabilization

doi: 10.1002/jcsm.70300

Figure Lengend Snippet: Effects of YOD1 silencing on DEX‐induced muscle atrophy in differentiated C2C12 myotubes. (a, b) C2C12 myotubes transfected with siRNA of each OTU family gene were treated with DEX for 48 h. Immunofluorescence (IF) was performed using an Alexa Fluor 488‐conjugated MYH antibody, and nuclei were stained with DAPI (a). Protein levels were determined using western blotting (b). (c–f) C2C12 myotubes transfected with control or YOD1 siRNA were treated with DEX for 48 h. Cells were fixed and stained with Giemsa (c). IF was performed using an Alexa Fluor 546‐conjugated MYH antibody, and nuclei were stained with DAPI (d). Protein (e) and mRNA (f) levels were determined using western blotting and qPCR, respectively. # p < 0.01 compared to the control. ** p < 0.01 compared to DEX.

Article Snippet: C2C12 myoblast cell line (CRL‐1772, ATCC, VA, USA) was cultured in growth medium (GM; DMEM‐H supplemented with 10% FBS) in a humidified atmosphere containing 5% CO 2 at 37°C.

Techniques: Transfection, Immunofluorescence, Staining, Western Blot, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Deubiquitinase YOD1 Inhibition Suppresses DEX‐ and Denervation‐Induced Muscle Atrophy Through MAFbx Destabilization

doi: 10.1002/jcsm.70300

Figure Lengend Snippet: YOD1 deubiquitinases and stabilizes MAFbx. (a) C2C12 myotubes were transfected with control or YOD1 siRNA and then treated with 20 μg/mL of cycloheximide (CHX) for the indicated durations. (b) C2C12 myotubes were transfected with control or YOD1 siRNA and treated with 0.25 μM of MG132, followed by DEX treatment for 12 h. (c) To analyse the ubiquitination of endogenous MAFbx, C2C12 myotubes were co‐transfected with control or YOD1 siRNA in the presence of HA‐Ub and treated with 0.25 μM of MG132, followed by DEX treatment for 24 h. Ubiquitination of endogenous MAFbx was detected using the ubiquitination assay. (d,e) C2C12 myotubes were transfected with vector, GFP‐YOD1 WT or GFP‐YOD1 C160S plasmid and then treated with DEX (d) or 20 μg/mL of CHX (e) for the indicated durations. (f) C2C12 myoblasts were co‐transfected with vector, GFP‐YOD1 WT or GFP‐YOD1 C160S plasmid in the presence of HA‐Ub and FLAG‐MAFbx and treated with MG132 for 12 h. Ubiquitination of exogenous MAFbx was detected using the ubiquitination assay. The band intensity of MAFbx was analysed using ImageJ.

Article Snippet: C2C12 myoblast cell line (CRL‐1772, ATCC, VA, USA) was cultured in growth medium (GM; DMEM‐H supplemented with 10% FBS) in a humidified atmosphere containing 5% CO 2 at 37°C.

Techniques: Transfection, Control, Ubiquitin Proteomics, Plasmid Preparation

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Deubiquitinase YOD1 Inhibition Suppresses DEX‐ and Denervation‐Induced Muscle Atrophy Through MAFbx Destabilization

doi: 10.1002/jcsm.70300

Figure Lengend Snippet: YOD1 interacts with MAFbx and removes polyubiquitin chains at K48 of MAFbx. (a) C2C12 myotubes were treated with or without DEX for 48 h. Cell lysates were immunoprecipitated with an anti‐MAFbx antibody, followed by immunoblotting (IB) with anti‐YOD1 or anti‐MAFbx antibodies. (b) C2C12 myoblasts were co‐transfected with vector, GFP‐YOD1 WT, or GFP‐YOD1 C160S in the presence of FLAG‐MAFbx. Interactions were demonstrated using IP. (c) C2C12 myoblasts were co‐transfected with vector, GFP‐YOD1 WT, GFP‐YOD1 ΔZn, or GFP‐YOD1 ΔUBX in the presence of FLAG‐MAFbx (left panel). C2C12 myoblasts were co‐transfected with vector, FLAG‐MAFbx WT, FLAG‐MAFbx ΔF‐box, FLAG‐MAFbx ΔNSL2, FLAG‐MAFbx ΔLZ, or FLAG‐MAFbx c‐terminal in the presence of GFP‐YOD1 WT (right panel). Interactions were demonstrated using IP. (d) C2C12 myoblasts were transfected with vector, FLAG‐MAFbx WT, FLAG‐MAFbx K29R, FLAG‐MAFbx K48R, or FLAG‐MAFbx K267R and then treated with 20 μg/mL of CHX for the indicated durations. The band intensity of FLAG was analysed using ImageJ. (e, f) C2C12 myoblasts were co‐transfected with vector, FLAG‐MAFbx WT, FLAG‐MAFbx K29R, FLAG‐MAFbx K48R or FLAG‐MAFbx K267R in the presence of control or YOD1 siRNA. The protein level (e) and ubiquitination of exogenous MAFbx (f) was measured using western blotting and ubiquitination assay, respectively.

Article Snippet: C2C12 myoblast cell line (CRL‐1772, ATCC, VA, USA) was cultured in growth medium (GM; DMEM‐H supplemented with 10% FBS) in a humidified atmosphere containing 5% CO 2 at 37°C.

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Control, Ubiquitin Proteomics

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Deubiquitinase YOD1 Inhibition Suppresses DEX‐ and Denervation‐Induced Muscle Atrophy Through MAFbx Destabilization

doi: 10.1002/jcsm.70300

Figure Lengend Snippet: Effect of G5 on DEX‐induced muscle atrophy in C2C12 myotubes. (a–d) C2C12 myotubes were treated with G5, followed by DEX for 48 h. The cells were fixed and stained with Giemsa stain (a). IF was performed using an Alexa Fluor 546‐conjugated MYH antibody, and nuclei were stained with DAPI (b). Protein (c) and mRNA (d) levels were determined using western blotting and qPCR, respectively. # p < 0.01 compared to control. * p < 0.05 compared to DEX.

Article Snippet: C2C12 myoblast cell line (CRL‐1772, ATCC, VA, USA) was cultured in growth medium (GM; DMEM‐H supplemented with 10% FBS) in a humidified atmosphere containing 5% CO 2 at 37°C.

Techniques: Staining, Giemsa Stain, Western Blot, Control