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: 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: 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

Journal: Research

Article Title: Doping-Engineered Proangiogenic Nanozymes Orchestrate Ischemic Tissue Regeneration via Cytoprotection and Revascularization

doi: 10.34133/research.1260

Figure Lengend Snippet: Cu-doped Prussian blue (CuPB) nanozymes protect C2C12 myoblasts and H9c2 cardiomyocytes from H 2 O 2 -induced oxidative injury. (A and B) Representative fluorescence images and quantification of intracellular reactive oxygen species (ROS) in H 2 O 2 -injured C2C12 cells after Prussian blue (PB) or CuPB treatment, detected using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Scale bar: 50 μm. n = 5. (C and D) Representative terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining images and quantification of apoptotic C2C12 cells following H 2 O 2 injury with PB or CuPB treatment. Scale bar: 50 μm. n = 5. (E) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of apoptosis-related genes ( Bcl2 , Caspase3 , Caspase9 , and Bax ) in C2C12 cells after different treatments. n = 3. (F and G) Representative fluorescence images and quantification of intracellular ROS in H 2 O 2 -injured H9c2 cells after PB or CuPB treatment, detected using the DCFH-DA probe. Scale bar: 50 μm. n = 5. (H and I) Representative TUNEL staining images and quantification of apoptotic H9c2 cells following H 2 O 2 injury with PB or CuPB treatment. Scale bar: 50 μm. n = 5. (J) qRT-PCR analysis of apoptosis-related gene expression ( Bcl2 , Caspase3 , Caspase9 , and Bax ) in H9c2 cells after different treatments. n = 5.

Article Snippet: The rat cardiomyocyte cell line (H9c2) was obtained from Procell Life Science & Technology Co., Ltd. (China), and the mouse myoblast cell line (C2C12) was purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd., the authorized American Type Culture Collection distributor in China (CRL1772).

Techniques: Fluorescence, End Labeling, TUNEL Assay, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression

Journal: Research

Article Title: Doping-Engineered Proangiogenic Nanozymes Orchestrate Ischemic Tissue Regeneration via Cytoprotection and Revascularization

doi: 10.34133/research.1260

Figure Lengend Snippet: Cu-doped Prussian blue (CuPB) nanozymes protect C2C12 myoblasts and H9c2 cardiomyocytes from H 2 O 2 -induced oxidative injury. (A and B) Representative fluorescence images and quantification of intracellular reactive oxygen species (ROS) in H 2 O 2 -injured C2C12 cells after Prussian blue (PB) or CuPB treatment, detected using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Scale bar: 50 μm. n = 5. (C and D) Representative terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining images and quantification of apoptotic C2C12 cells following H 2 O 2 injury with PB or CuPB treatment. Scale bar: 50 μm. n = 5. (E) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of apoptosis-related genes ( Bcl2 , Caspase3 , Caspase9 , and Bax ) in C2C12 cells after different treatments. n = 3. (F and G) Representative fluorescence images and quantification of intracellular ROS in H 2 O 2 -injured H9c2 cells after PB or CuPB treatment, detected using the DCFH-DA probe. Scale bar: 50 μm. n = 5. (H and I) Representative TUNEL staining images and quantification of apoptotic H9c2 cells following H 2 O 2 injury with PB or CuPB treatment. Scale bar: 50 μm. n = 5. (J) qRT-PCR analysis of apoptosis-related gene expression ( Bcl2 , Caspase3 , Caspase9 , and Bax ) in H9c2 cells after different treatments. n = 5.

Article Snippet: The rat cardiomyocyte cell line (H9c2) was obtained from Procell Life Science & Technology Co., Ltd. (China), and the mouse myoblast cell line (C2C12) was purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd., the authorized American Type Culture Collection distributor in China (CRL1772).

Techniques: Fluorescence, End Labeling, TUNEL Assay, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression