murine myoblast cells c2c12 human prostate adenocarcinoma cell lines  (ATCC)

Journal: bioRxiv

Article Title: Pancreatic cancer extracellular vesicles carry a time-of-day-regulated miRNA cargo that disrupts the skeletal muscle clock and bioenergetics

doi: 10.64898/2026.05.03.722338

Figure Lengend Snippet: (A) Bioluminescence recording, ( B ) period analysis, and ( C ) phase and amplitude analysis of U2OS BMAL1 :Luc reporter cells treated with PANC-1 CM at 12.5%, 25%, 50%, and 100% of the recording media. ( D ) Bioluminescence recording, ( E ) period analysis, and ( F ) phase and amplitude analysis of NIH3T3 Bmal1 :Luc reporter cells treated with PANC-1 CM at the same concentrations. For all bioluminescence experiments, at least three complete oscillations were included in the period estimation, excluding the first 24 h. Mean ± SD of relative mRNA expression of NIH3T3 core clock genes Bmal1 ( G ), Per2 ( H ), and Cry2 ( I ) measured over 36 h in response to PANC-1 CM. Relative mRNA levels of core clock genes in synchronized C2C12 myotubes over 32 h following treatment with PANC-1 CM: ( J ) Bmal1 , ( K ) Per2 , and ( L ) Cry2 . Cosine curves were fit for visualization purposes only; solid lines represent rhythmic oscillations (p<0.05) detected by MetaCycle, while dashed lines indicate loss of statistical rhythmicity (Suppl. Table 1). ( G–L ) Black: Control; ( G–I ) Red: PANC-1 CM; ( J–L ) Blue: PANC-1 CM. ( M ) Schematic representation and representative images of mature C2C12 myotube atrophy in response to NIH3T3 or PANC-1 released factors using a Transwell co-culture system; three measurements per myotube (yellow arrows) were used to quantify shortening. ( N ) Quantification of normalized myotube diameter under NIH3T3 vs PANC-1 co-culture, normalized to NIH3T3 co-culture control. One-way ANOVA: ( B ) p=0.0009, ( E ) p=0.0087. ( B, E ) Dunnett’s post-hoc test: *p<0.05; **p<0.01; ***p<0.001. (N) Student’s t-test: ***p<0.001.

Article Snippet: The human pancreatic cancer cell line PANC-1, the murine fibroblast cell line NIH3T3, and the murine myoblast cell line C2C12 were purchased from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Control, Co-Culture Assay

Journal: bioRxiv

Article Title: Pancreatic cancer extracellular vesicles carry a time-of-day-regulated miRNA cargo that disrupts the skeletal muscle clock and bioenergetics

doi: 10.64898/2026.05.03.722338

Figure Lengend Snippet: (A) Top 35 miRNAs by mean expression in PANC-1-derived sEVs. Bar plot of mean log2 CPM across 9 time-points (4–36 h). Red bars: miRNAs selected from the top 35 to be tested in the BMAL1 :Luc reporter and atrophy assays; grey bars: remaining top-35 miRNAs. miRNAs are ranked in descending order of EV expression. ( B ) GO Biological Process enrichment of the experimentally validated targets (miRTarBase) of the 11 selected miRNAs. Terms are grouped into functional categories. Dot size represents the number of validated target genes associated with each term; dot color indicates Gene Ratio (proportion of input genes annotated to the term), from light pink (low) to dark red (high). Analysis performed with clusterProfiler. ( C , top panel) Normalized C2C12 myotube diameter at 0, 24, and 48 h post-transfection with miR-27b-3p, miR-615-3p, miR-191-5p, miR-127-3p, miR-99b-5p, or negative-transfection control (NTC); dexamethasone (Dexa) included as positive control. ( C , lower panel) Normalized C2C12 myotube diameter at the same time-points after transfection with hsa-let-7f-5p, miR-183-5p, miR-92a-3p, miR-30c-5p, miR-26a-5p, miR-10a-5p, NTC, or Dexa. ( D ) Oxygen consumption rate (OCR; top), resting-phenotype plot of basal OCR vs ECAR (middle), and metabolic-capacity plot of maximal OCR vs ECAR following FCCP (lower) for mature C2C12 myotubes 48 h post-transfection with miR-27b-3p, miR-615-3p, miR-191-5p, or NTC (Control). ( E ) Same panels for myotubes transfected with miR-127-3p, miR-99b-5p, miR-183-5p, or NTC. Sequential injections of oligomycin, FCCP, and rotenone/antimycin A were used to dissect mitochondrial respiration. Data are presented as mean ± SEM. ( C ) Measurements were taken from at least 5 random fields per well in N=3 wells; statistical analysis used 2-way ANOVA with Dunnett’s post-hoc correction: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: The human pancreatic cancer cell line PANC-1, the murine fibroblast cell line NIH3T3, and the murine myoblast cell line C2C12 were purchased from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Derivative Assay, Functional Assay, Transfection, Control, Positive 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: 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