Journal: Cancer Discovery

Article Title: CD74–NRG1 Fusions in Lung Adenocarcinoma

doi: 10.1158/2159-8290.cd-13-0633

Figure Lengend Snippet: Figure 1. Identifi cation of the CD74–NRG1 fusion gene. A, overview of driver genes detected in a cohort of 25 EGFR - and KRAS -negative lung adenocarcinomas of never smokers. B, detection of CD74–NRG1 fusion transcript by transcriptome sequencing. Schematic representation of the fusion transcript domains and some of the transcriptome sequencing reads spanning the fusion point. C, expression levels of NRG1 isoforms in 15 unselected and 23 pan-negative lung adenocarcinomas (AD; wild-type for EGFR , KRAS , BRAF , ERBB2 , ALK , ROS , and RET ), and, in the index case, inferred from tran- scriptome sequencing data. Average FPKM values are shown (top). RNAseq analysis for NRG1 reads to show where the breakpoint of CD74–NRG1 occurs. The dip in exon 4 represents reads of the fusion that could not be mapped. No reads could be mapped to exons 1–3 (bottom). D, top, the genomic intron/ exon structure of the CD74 (in green) and the NRG1 locus (in orange) with the genomic breakpoints marked in red. Sequencing reads were obtained from hybrid-capture–based genomic sequencing of 333 genes using genomic DNA of the index case (see Methods). The breakpoint-spanning reads are shown by means of the Integrative Genomics Viewer ( www.broadinstitute.org/igv/ ) focused on the CD74 gene (bottom). The gray area of the read is aligned to the CD74 reference sequence. Colored area on the right indicates bases not matching the CD74 reference sequence. Sequence comparison reveals alignment to the NRG1 reference sequence. Encompassing reads whose mate pairs are mapped to the NRG1 locus on chromosome 8 are displayed in dark purple. Bottom, a representative picture of NRG1 break-apart FISH. Arrows, break-apart signals.

Article Snippet: Anti-human CD74 was obtained from Abcam (Catalog No. # ab22603), and anti-polyclonal NRG1 β 1 was obtained from R&D Systems (Catalog No. AF396-NA).

Techniques: Sequencing, Expressing, Genomic Sequencing, Comparison

Journal: Cancer Discovery

Article Title: CD74–NRG1 Fusions in Lung Adenocarcinoma

doi: 10.1158/2159-8290.cd-13-0633

Figure Lengend Snippet: Figure 3. Functional relevance of CD74–NRG1. A, expression levels of ERBB receptors in the index case inferred from transcriptome sequencing data. FPKM values are shown. B, levels of p-ERBB2 and p-ERBB3 detected by immunohistochemical analysis in a CD74–NRG1-positive case using and antibody directed against ERBB2 Tyr1221/1222 and ERBB2 Tyr1289. C, the same p-ERBB3 antibody was used to stain a tissue microarray composed of 241 lung adenocarcinomas. The frequency of p-ERBB3–positive cases in this cohort versus the fi ve CD74–NRG1-positive samples is shown ( P < 0.0001). D, activation of the PI3K–AKT pathway detected by Western blot analysis of H322 and H1568 lung cancer cells transduced with retroviruses encoding CD74–NRG1 or the empty vector control (e.v.). E, levels of p-ERBB3 and p-AKT measured by Western blot analysis in the presence of an empty vector, CD74–NRG1, or a truncated version lacking the EGF-like domain (CD74–NRG1_ΔEGF). F, anchorage-independent growth of H1568 cells expressing an empty vector, CD74–NRG1, or a truncated version lacking the EGF-like domain (CD74–NRG1_ΔEGF). Top, the average colony size for the three condi- tions, with error bars representing standard deviations. The experiment was performed with two independent transductions for a total of four times. **, P < 0.01; ***, P < 0.001. Bottom, representative pictures of the colony formation assay. Please note that H1568 cells are oncogenic and form small colonies without any manipulation.

Article Snippet: Anti-human CD74 was obtained from Abcam (Catalog No. # ab22603), and anti-polyclonal NRG1 β 1 was obtained from R&D Systems (Catalog No. AF396-NA).

Techniques: Functional Assay, Expressing, Sequencing, Immunohistochemical staining, Staining, Microarray, Activation Assay, Western Blot, Transduction, Plasmid Preparation, Control, Colony Assay

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: International journal of oncology

Article Title: Androgen-independent prostate cancer cells circumvent EGFR inhibition by overexpression of alternative HER receptors and ligands.

doi: 10.3892/ijo.2012.1509

Figure Lengend Snippet: Figure 1. Gene expression pattern of HER family members in DU145 and PC3 cells. The basal mRNA level of each HER receptor (EGFR, HER2, HER3, HER4) and ligand (EGF, epidermal growth factor; TGF-α, transforming growth factor-α; AR, amphiregulin; BTC, betacellulin; EPR, epiregulin; NRG-1, neu regulin-1; HB-EGF, heparin-binding EGF) was assessed by real-time PCR. Each value was normalized versus the corresponding mRNA level of the TATA box binding protein (TBP) constitutive gene. The bars represent the mean value ± SE of three independent quantifications.

Article Snippet: The mouse monoclonal antibody against NRG-1β was from R&D Systems (Minneapolis, MN, USA).

Techniques: Gene Expression, Binding Assay, Real-time Polymerase Chain Reaction

Journal: International journal of oncology

Article Title: Androgen-independent prostate cancer cells circumvent EGFR inhibition by overexpression of alternative HER receptors and ligands.

doi: 10.3892/ijo.2012.1509

Figure Lengend Snippet: Figure 4. Changes in the gene expression of HER receptors and ligands after EGFR inhibition. (A) DU145 and (B) PC3 cells were treated with either the corre sponding IC30 of cetuximab (350 µg/ml and 500 µg/ml, respectively), the corresponding IC50 of erlotinib (2.5 µM and 15 µM, respectively) and gefitinib (2.5 µM and 15 µM, respectively) or vehicle as a control. After 24 h, the mRNA level of each HER receptor (EGFR, HER2, HER3, HER4) and ligand (EGF, epidermal growth factor; TGF-α, transforming growth factor-α; AR, amphiregulin; BTC, betacellulin; EPR, epiregulin; NRG-1, neuregulin-1; HB-EGF, heparin-binding EGF) was assessed by real-time PCR in each cell line and values were normalized against the corresponding mRNA expression of the TBP constitutive gene. Then, the change in the expression of each particular gene induced by the treatments was determined by comparing the normalized values in treated cells against the cor responding normalized value in the control cells. The bars indicate the mean fold change ± SE of three independent quantifications (*P<0.05 vs. control cells). Bars over the dotted line indicate an increase in the gene expression compared to the control cells, while bars under the dotted line represent impaired gene expression after the treatment.

Article Snippet: The mouse monoclonal antibody against NRG-1β was from R&D Systems (Minneapolis, MN, USA).

Techniques: Gene Expression, Inhibition, Control, Binding Assay, Real-time Polymerase Chain Reaction, Expressing