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: 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