Journal: Burns & Trauma

Article Title: Enhancing diabetic muscle repair through W-GA nanodots: a nanomedicinal approach to ameliorate myopathy in type 2 diabetes

doi: 10.1093/burnst/tkae059

Figure Lengend Snippet: Evaluation of the biocompatibility of W-GA in vitro. ( a ) C2C12 cells cultured for 7 days in W-GA were evaluated for proliferation and cytotoxicity using live/dead staining. Scale bar = 500 μm. ( b ) Survival rate of C212 cells ( n = 3) is shown as the mean ± standard deviation. The statistical significance of differences between treatments was determined by one-way ANOVA and the Bonferroni posthoc correction. NS: Not significant. ( c ) C2C12 cell proliferation capacity was assessed 24 hours post-treatment using BrdU incorporation. The green signal represents BrdU. Scale bar = 500 μm. ( d ) Quantification of BrdU assay data ( n = 3). The data are presented as the mean ± standard deviation. NS: Not significant. ( e ) The cell proliferation ability of C2C12 cells in the W-GA group was further evaluated using a CCK-8 assay. The data are presented as the mean ± standard deviation. NS: Not significant

Article Snippet: C2C12 myoblasts (a mouse cell line) were obtained from ScienCell Research Laboratories and cultured in Dulbecco's modified Eagle’s medium (DMEM; containing 25 mM glucose; Gibco, USA), which included 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA), in a humidified atmosphere at 37°C and 5% CO 2 .

Techniques: In Vitro, Cell Culture, Staining, Standard Deviation, BrdU Incorporation Assay, BrdU Staining, CCK-8 Assay

Journal: Burns & Trauma

Article Title: Enhancing diabetic muscle repair through W-GA nanodots: a nanomedicinal approach to ameliorate myopathy in type 2 diabetes

doi: 10.1093/burnst/tkae059

Figure Lengend Snippet: Antiapoptotic, antioxidative, and myogenic differentiation-promoting effects of W-GA. ( a ) Flow cytometry profiles showing the abundance of total C2C12 cells under various treatment conditions, along with apoptosis events in C2C12 cells under different therapeutic interventions. ( b ) Quantification of flow cytometry data for apoptotic cells ( n = 3). The data are presented as the mean ± standard deviation. NS: Not significant, * * p < 0.01. The statistical significance of differences between treatments was determined by one-way ANOVA and the Bonferroni posthoc correction. ( c ) Flow cytometry profiles showing the production of ROS in C2C12 cells under different treatment conditions. ( d ) Quantification of flow cytometry data for ROS production ( n = 3). The data are presented as the mean ± standard deviation. NS: Not significant, * p < 0.05. ( e ) Representative immunofluorescence image illustrating MYHC and MyoD protein expression in C2C12 myoblasts. Scale bar = 100 μm. ( f ) Quantitative analysis and intergroup comparison of myotube diameters ( n = 3). The statistical significance of differences between treatments was determined by one-way ANOVA and the Bonferroni posthoc correction. NS: Not significant, * p < 0.05, * * p < 0.01

Article Snippet: C2C12 myoblasts (a mouse cell line) were obtained from ScienCell Research Laboratories and cultured in Dulbecco's modified Eagle’s medium (DMEM; containing 25 mM glucose; Gibco, USA), which included 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA), in a humidified atmosphere at 37°C and 5% CO 2 .

Techniques: Flow Cytometry, Standard Deviation, Immunofluorescence, Expressing, Comparison

Journal: Cellular and Molecular Life Sciences

Article Title: Notch1 signaling is mediated by importins alpha 3, 4, and 7

doi: 10.1007/s00018-010-0378-7

Figure Lengend Snippet: NICD in vitro binds to importins α3, α4, and α7. a GST pull-down assays were performed with lysate of HEK293 cells stably transfected with NotchΔE and purified recombinant GST-importins as indicated. Proteins were separated on SDS-PAGE, blotted and labeled with NICD-specific antibody ( top ) or stained with Coomassie Brilliant Blue ( bottom ). b Using purified recombinant GST-NICD, pull-down assays were performed from lysates of C2C12 cells ( left ) or mouse skeletal muscle ( right ). Lysates, pull-down, and as controls pull-down with GST protein and GSH-beads were separated on SDS-PAGE, blotted, and labeled with importin-specific antibodies as indicated ( top , WB) or gels were stained with Coomassie Brilliant Blue ( bottom ). Asterisk , unspecific bands; WB , Western blot. c For co-immunoprecipitation experiment (Co-IP) C2C12 cell lysate transiently transfected with NICD-myc was immunoprecipitated with anti-importin α4 antibody or normal goat immunoglobulins. Lysate and Co-IPs were separated on SDS-PAGE, blotted, and labeled with importin α4- and myc-specific antibodies

Article Snippet: HeLa Kyoto cells and mouse myoblast C2C12 cells were kindly provided by Rainer Pepperkok (EMBL, Heidelberg) and Rüdiger Rudolf (Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen), respectively.

Techniques: In Vitro, Stable Transfection, Transfection, Purification, Recombinant, SDS Page, Labeling, Staining, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Notch1 signaling is mediated by importins alpha 3, 4, and 7

doi: 10.1007/s00018-010-0378-7

Figure Lengend Snippet: Endogenous Notch signaling in myoblasts is mainly mediated by importins α3 and α4. a C2C12 cells transfected with siRNAs against importin isoforms as indicated were transfected with or without Delta1 cDNA and Notch reporter construct. The γ-secretase inhibitor DAPT was used to show γ-secretase dependency of the measured Notch activity. Firefly/renilla activities were determined and the activity in Delta1 transfected cells set to 100%. Means ± SD of five independent experiments are shown. Asterisks indicate significance ( p < 0.05, Student's t test). b Western-blot analysis of importin KD efficiency and specificity. C2C12 cell lysates were separated on SDS-PAGE, blotted, and probed with antibodies as indicated. c C2C12 cells were transfected with control (ctrl) siRNA or pooled siRNAs against importins α3, α4, and α7 and subsequently with or without Delta1 as indicated. Where indicated, cells were incubated with DAPT for 24 h. After RNA isolation, quantitative real-time PCR for Hey1 expression was performed. Hey1 expression level after Delta1 induction was set to 100% and the other values related to that. Means ± SD of three independent experiments are shown

Article Snippet: HeLa Kyoto cells and mouse myoblast C2C12 cells were kindly provided by Rainer Pepperkok (EMBL, Heidelberg) and Rüdiger Rudolf (Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen), respectively.

Techniques: Transfection, Construct, Activity Assay, Western Blot, SDS Page, Control, Incubation, Isolation, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Biomedical Science

Article Title: Vitamin D3/VDR resists diet-induced obesity by modulating UCP3 expression in muscles

doi: 10.1186/s12929-016-0271-2

Figure Lengend Snippet: Calcitriol enhanced relative luciferase activity in a dose dependent manner. UCP3-pro-luc was transfected into C2C12 cells ( a ), L6 cells ( b ), and H-EMC-SS cells ( c ). Relative luciferase activity was analyzed by Dual Luciferase Assay after stimulation with calcitriol. n = 3, compared with vehicle, *: P < 0.05; **: P < 0.01

Article Snippet: Mouse C2C12 muscle cells (C2C12), rat L6 muscle cells (L6), H-EMC-SS chondrosarcoma cells, and human embryonic kidney cells (HEK293) were purchased from JCRB Cell Bank (National Institute of Biomedical Innovation, Tokyo, Japan).

Techniques: Luciferase, Activity Assay, Transfection

Journal: Journal of Biomedical Science

Article Title: Vitamin D3/VDR resists diet-induced obesity by modulating UCP3 expression in muscles

doi: 10.1186/s12929-016-0271-2

Figure Lengend Snippet: Calcitriol increased UCP3 mRNA expression in muscle cells. Relative mRNA expression was detected in C2C12 cells ( a ), L6 cells ( b ), and H-EMC-SS cells ( c ) using qRT-PCR. To investigate the role of calcitriol and VDR on UCP3 mRNA expression, we transfected with a control plasmid ((-) mock) or VDR expression plasmid ((+) pCMX-VDR). We also performed loss-of-function experiments using selective siRNA against UCP3 ( a ). Compared with non-transfected controls, siRNAs against UCP3 strongly reduced the expression of the UCP3. n = 3. Compared with vehicle, *; P < 0.05, **; P < 0.01. ***; P < 0.005

Article Snippet: Mouse C2C12 muscle cells (C2C12), rat L6 muscle cells (L6), H-EMC-SS chondrosarcoma cells, and human embryonic kidney cells (HEK293) were purchased from JCRB Cell Bank (National Institute of Biomedical Innovation, Tokyo, Japan).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation

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