Journal: Scientific Reports

Article Title: Modulation of the Neuregulin 1/ErbB system after skeletal muscle denervation and reinnervation

doi: 10.1038/s41598-018-23454-8

Figure Lengend Snippet: The expression of ErbB receptors and NRG1 isoforms is modulated in muscle after nerve injury. Three different types of nerve injury and injury/repair were performed on the median nerve (denervation, crush and end-to-end repair). At different time points, SDF muscle was collected and used for expression analysis. Graphs show the results of quantitative real-time PCR analysis. The analysis was performed on three animals per group; data are expressed as the mean ± SEM. Statistical analysis comparing groups at each single time point: one-way ANOVA plus Bonferroni’s post hoc test: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. E-E: end-to-end repair.

Article Snippet: For the molecular analysis, differentiated cells were treated as follows: 1 μM dexamethasone (D2915, Sigma) was added to the differentiation medium, alone or in combination with recombinant peptides corresponding to the EGF-like domain of NRG1α (30 nM; #296-HR) or NRG1β (5 nM, #396-HB) purchased from R&D systems, for 24 h. For myotube diameter analysis, cells were treated for 24 h with 1 μM dexamethasone, followed by 48 h treatment with dexamethasone alone or in combination with NRG1α or NRG1β.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Modulation of the Neuregulin 1/ErbB system after skeletal muscle denervation and reinnervation

doi: 10.1038/s41598-018-23454-8

Figure Lengend Snippet: NRG1/ErbB expression after 12 weeks of muscle denervation and reinnervation. Twelve weeks after denervation or end-to-end repair (E-E), the expression of ErbB receptors and NRG1 was analysed by quantitative real-time PCR in SDF muscle. The analysis was performed on three animals per group; data are expressed as a mean ± SEM. The significant difference between the sample and the control is indicated over the sample bar. Statistical analysis: one-way ANOVA plus Bonferroni’s post hoc test: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Article Snippet: For the molecular analysis, differentiated cells were treated as follows: 1 μM dexamethasone (D2915, Sigma) was added to the differentiation medium, alone or in combination with recombinant peptides corresponding to the EGF-like domain of NRG1α (30 nM; #296-HR) or NRG1β (5 nM, #396-HB) purchased from R&D systems, for 24 h. For myotube diameter analysis, cells were treated for 24 h with 1 μM dexamethasone, followed by 48 h treatment with dexamethasone alone or in combination with NRG1α or NRG1β.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: Modulation of the Neuregulin 1/ErbB system after skeletal muscle denervation and reinnervation

doi: 10.1038/s41598-018-23454-8

Figure Lengend Snippet: Dexamethasone-treated C2C12 myotubes as an in vitro model to study the NRG1/ErbB system during muscle atrophy. Twenty-four hours after 1 µM dexamethasone treatment, alone or in combination with 30 nM NRG1α or 5 nM NRG1β, C2C12 mRNA was extracted for quantitative real-time PCR analysis. Data refer to biological triplicates and are expressed as the mean ± SD. The significant difference between a sample and the control is indicated over the sample bar. Statistical analysis: one-way ANOVA plus Bonferroni’s post hoc test: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. DEXA: dexamethasone.

Article Snippet: For the molecular analysis, differentiated cells were treated as follows: 1 μM dexamethasone (D2915, Sigma) was added to the differentiation medium, alone or in combination with recombinant peptides corresponding to the EGF-like domain of NRG1α (30 nM; #296-HR) or NRG1β (5 nM, #396-HB) purchased from R&D systems, for 24 h. For myotube diameter analysis, cells were treated for 24 h with 1 μM dexamethasone, followed by 48 h treatment with dexamethasone alone or in combination with NRG1α or NRG1β.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: Modulation of the Neuregulin 1/ErbB system after skeletal muscle denervation and reinnervation

doi: 10.1038/s41598-018-23454-8

Figure Lengend Snippet: Molecular and morphological analysis of dexamethasone-induced atrophy in C2C12 myotubes treated with NRG1. ( a ) Representative pictures of western blot assays performed with proteins extracted from C2C12 myotubes treated for 24 h with 1 µM dexamethasone alone or in combination with 30 nM NRG1α or 5 nM NRG1β. ( b ) C2C12 myotube protein quantification. ( c ) Myotube diameter measurement. C2C12 cells were treated for 24 h with dexamethasone and then for 48 h with dexamethasone alone or in combination with NRG1α or NRG1β. The significant difference between a sample and the control is indicated over the sample bar. ( d ) Representative images used for myotube diameter measurement. Data refer to a biological triplicate and are expressed as the mean ± SD. Statistical analysis: one-way ANOVA plus Bonferroni’s post hoc test: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. DEXA: dexamethasone.

Article Snippet: For the molecular analysis, differentiated cells were treated as follows: 1 μM dexamethasone (D2915, Sigma) was added to the differentiation medium, alone or in combination with recombinant peptides corresponding to the EGF-like domain of NRG1α (30 nM; #296-HR) or NRG1β (5 nM, #396-HB) purchased from R&D systems, for 24 h. For myotube diameter analysis, cells were treated for 24 h with 1 μM dexamethasone, followed by 48 h treatment with dexamethasone alone or in combination with NRG1α or NRG1β.

Techniques: Western Blot, Control

Journal:

Article Title: Role of proneuregulin 1 cleavage and human epidermal growth factor receptor activation in hypertonic aquaporin induction

doi: 10.1073/pnas.0406853101

Figure Lengend Snippet: HER2 and HER3 are activated in MLE-12 cells. (A) To examine cells for activation of HER2 and HER3, exogenous NRG1-β (10 ng/ml) was added to the medium, and cells were harvested at the designated times for immunoprecipitation (IP) of HER2 or HER3. Immunoprecipitates were probed for total HER2 or HER3, phosphotyrosine (P-tyrosine), and coimmunoprecipitation of adaptor molecules p85α (PI3-kinase subunit) and Shc. An aliquot of the cell lysate was probed for P-ERK or total ERK. Within 1 min, NRG1-β increased tyrosine phosphorylation of HER2, Shc binding to HER2, and p85α and Shc binding to HER3. Addition of NRG1-β led to ERK phosphorylation within 1 min that was maximal at 5 min. (B) To determine whether hypertonic stress activated HER2 or HER3, hypertonic medium was added and cells were harvested at the designated times for analysis as in A. Consistent with the effects of exogenous NRG1-β, hypertonic shock increased tyrosine phosphorylation of HER2, Shc binding to HER2, and p85α and Shc binding to HER3 within 1–3 min. P-ERK was detectable by 1 min and increased to 10 min.

Article Snippet: The following antibodies were used: anti-total and antiphosphorylated ERK from New England Biolabs; anti-EGF receptor isoforms 1–4 (HER1–4), anti-NRG1-precursor -α and -β, anti-phosphatidylinositol 3-kinase (PI3-kinase) subunit p85α, anti-growth factor receptor-bound protein 2 (GRB2), and anti-GRB7 from Santa Cruz Biotechnology; anti-phosphotyrosine from Upstate Biotechnology (Lake Placid, NY); anti-Shc from BD Transduction Laboratories (San Diego); anti-AQP3 from Alpha Diagnostics (San Antonio, TX); neutralizing antibodies to NRG1-α and NRG1-β and recombinant NRG1-α and NRG1-β (EGF domain) from R & D Systems; and horseradish peroxidase-coupled secondary antibodies from Amersham Pharmacia Biotech (mouse and rabbit) and Santa Cruz Biotechnology (goat).

Techniques: Activation Assay, Immunoprecipitation, Binding Assay