Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Neuregulin-1 is a chemoattractant and chemokinetic molecule for trunk neural crest cells

doi: 10.1002/dvdy.24625

Figure Lengend Snippet: (A) Isolated TNCCs plated on Boyden inserts in the presence of different molecular gradients. Data is internally normalized to control. All gradients tested except NGF were significantly different than control (p<0.05 by paired t-test; n=circles on graph/experimental dates). GDNF, glial cell-derived neurotrophic factor. Red line, 100% of control. (B) Isolated TNCC plated on FN slides were filmed with or without 200ng/ml of NRG1. Total migrated distance and Euclidean distance were normalized showing increased migration after NRG1 treatment (p<0.07 and p<0.02 respectively. N=3 experiments, N of cells =86). (C) Velocity of cells in B was measured, with NRG1 treated cells moving twice as fast as control cells (p<0.017). (D–I) NT explants cultured in Boyden chambers with an NRG1β gradient (−/+), no NRG1β (−/−), or homogenous NRG1β (+/+). (D–G) Representative HNK1 staining of chemotaxis filters showing a higher percent TNCCs on the bottom side in −/+ versus −/− chambers. Top of filter in focus (D,E); bottom in focus (F,G). Bar, 25 μm. (H) Percent HNK1-positive and percent HNK1-negative transmigrated cells. n=7 experimental dates (>33 NT explants per treatment). p<0.0001 for HNK1-positive −/− versus −/+ treatments, 1-tailed Dunnett’s. (I) Combined TNCC density from top and bottom sample areas. All t-tests described: no multiple testing adjustments made. Bar graphs show means +/− SEM.

Article Snippet: Amplification was done over 35 cycles (95°C 5minutes, 94°C 30 seconds, 58°C 30 seconds, 72°C 1 minute) for RT-PCR (Eppendorf master cycler gradient). table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse HNK-1 IgM DSHB Cat#1C10 Alexa 488 anti-mouse IgM Invitrogen Cat# A-21042 Mouse anti-c-erbB-4 IgG1 Thermo Scientific Cat#MS270P0 Rabbit anti-GFP IgG Life Technologies Cat# A-11122 Rhodamine Red-X anti-mouse IgM Jackson ImmunoResearch, West Grove, PA Cat#115-295-075 594 F(ab′)2 fragment of anti-mouse IgG ThermoFisher Cat#A-11020 488 anti-rabbit IgG ThermoFisher Cat#A-11034 594 anti-rabbit IgG ThermoFisher Cat# {"type":"entrez-nucleotide","attrs":{"text":"A11037","term_id":"492397","term_text":"A11037"}} A11037 647 anti-mouse IgM ThermoFisher Cat#A-21238 Bacterial and Virus Strains pcDNA3-DNerbB4 Rio et al. 1997 pcDNA3-EGFP Addgene #13031 Biological Samples Chicken embryos (Gallus gallus) AA Laboratories, Westminster, CA Quail embryos (Coturnix coturnix) McIntyre Poultry, Lakeside CA Chemicals, Peptides, and Recombinant Proteins Children’s Oncology Group Cell Culture and Xenograft Repository http://cogcell.org/ NRG1-EGF Domains NRG1α R&D Systems Cat#296-HR-050 BSA ThermoFisher Cat#BP-1600-100 NRG1β R&D Systems Cat#396-HB-050/CF Tryphostin AG 1478 Enzo Cat #ALX-270-036-M001 BPE BD Cat# 356123 Live Cell Imaging Solution Life Technologies Cat# A14291DJ Critical Commercial Assays Boyden Chamber Assay BD Immulon 4 ELISA well Dynatech Laboratories, Chantilly, VA microchips Caltech Microfluidic Foundry www.kni.caltech.edu/foundry Modified Zigmond Chamber Assay Walheim et al., 2012 Deposited Data Experimental Models: Cell Lines Experimental Models: Organisms/Strains Oligonucleotides Recombinant DNA Software and Algorithms Excel Microsoft https://products.office.com/en-us/excel SPSS IBM https://www.ibm.com/us-en/marketplace/spss-statistics ImageJ Manual Tracking ImageJ https://imagej.nih.gov/ij/plugins/track/track.html ImageJ Chemotaxis and Migration Tool ImageJ http://ibidi.com/software/chemotaxis_and_migration_tool AxioVision Rel.

Techniques: Isolation, Control, Derivative Assay, Migration, Cell Culture, Staining, Chemotaxis Assay

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Neuregulin-1 is a chemoattractant and chemokinetic molecule for trunk neural crest cells

doi: 10.1002/dvdy.24625

Figure Lengend Snippet: A NT explant (darker tissue) was plated within a region of an ELISA well pre-coated with NRG1α and filmed for 18 h as TNCCs emigrated from the NT explant and were confronted by the coat border (red curved line). A–C: Numerous emigrating cells can be seen migrating freely outside a PBS area (arrows). D–F: Numerous emigrating cells can be seen excessively lingering near the NRG1α border and/or preferentially migrating to open spaces still within the NRG1-coated region not occupied by other cells before migrating outside the coated region (arrows).

Article Snippet: Amplification was done over 35 cycles (95°C 5minutes, 94°C 30 seconds, 58°C 30 seconds, 72°C 1 minute) for RT-PCR (Eppendorf master cycler gradient). table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse HNK-1 IgM DSHB Cat#1C10 Alexa 488 anti-mouse IgM Invitrogen Cat# A-21042 Mouse anti-c-erbB-4 IgG1 Thermo Scientific Cat#MS270P0 Rabbit anti-GFP IgG Life Technologies Cat# A-11122 Rhodamine Red-X anti-mouse IgM Jackson ImmunoResearch, West Grove, PA Cat#115-295-075 594 F(ab′)2 fragment of anti-mouse IgG ThermoFisher Cat#A-11020 488 anti-rabbit IgG ThermoFisher Cat#A-11034 594 anti-rabbit IgG ThermoFisher Cat# {"type":"entrez-nucleotide","attrs":{"text":"A11037","term_id":"492397","term_text":"A11037"}} A11037 647 anti-mouse IgM ThermoFisher Cat#A-21238 Bacterial and Virus Strains pcDNA3-DNerbB4 Rio et al. 1997 pcDNA3-EGFP Addgene #13031 Biological Samples Chicken embryos (Gallus gallus) AA Laboratories, Westminster, CA Quail embryos (Coturnix coturnix) McIntyre Poultry, Lakeside CA Chemicals, Peptides, and Recombinant Proteins Children’s Oncology Group Cell Culture and Xenograft Repository http://cogcell.org/ NRG1-EGF Domains NRG1α R&D Systems Cat#296-HR-050 BSA ThermoFisher Cat#BP-1600-100 NRG1β R&D Systems Cat#396-HB-050/CF Tryphostin AG 1478 Enzo Cat #ALX-270-036-M001 BPE BD Cat# 356123 Live Cell Imaging Solution Life Technologies Cat# A14291DJ Critical Commercial Assays Boyden Chamber Assay BD Immulon 4 ELISA well Dynatech Laboratories, Chantilly, VA microchips Caltech Microfluidic Foundry www.kni.caltech.edu/foundry Modified Zigmond Chamber Assay Walheim et al., 2012 Deposited Data Experimental Models: Cell Lines Experimental Models: Organisms/Strains Oligonucleotides Recombinant DNA Software and Algorithms Excel Microsoft https://products.office.com/en-us/excel SPSS IBM https://www.ibm.com/us-en/marketplace/spss-statistics ImageJ Manual Tracking ImageJ https://imagej.nih.gov/ij/plugins/track/track.html ImageJ Chemotaxis and Migration Tool ImageJ http://ibidi.com/software/chemotaxis_and_migration_tool AxioVision Rel.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Neuregulin-1 is a chemoattractant and chemokinetic molecule for trunk neural crest cells

doi: 10.1002/dvdy.24625

Figure Lengend Snippet: RT-PCR analysis

Article Snippet: Amplification was done over 35 cycles (95°C 5minutes, 94°C 30 seconds, 58°C 30 seconds, 72°C 1 minute) for RT-PCR (Eppendorf master cycler gradient). table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse HNK-1 IgM DSHB Cat#1C10 Alexa 488 anti-mouse IgM Invitrogen Cat# A-21042 Mouse anti-c-erbB-4 IgG1 Thermo Scientific Cat#MS270P0 Rabbit anti-GFP IgG Life Technologies Cat# A-11122 Rhodamine Red-X anti-mouse IgM Jackson ImmunoResearch, West Grove, PA Cat#115-295-075 594 F(ab′)2 fragment of anti-mouse IgG ThermoFisher Cat#A-11020 488 anti-rabbit IgG ThermoFisher Cat#A-11034 594 anti-rabbit IgG ThermoFisher Cat# {"type":"entrez-nucleotide","attrs":{"text":"A11037","term_id":"492397","term_text":"A11037"}} A11037 647 anti-mouse IgM ThermoFisher Cat#A-21238 Bacterial and Virus Strains pcDNA3-DNerbB4 Rio et al. 1997 pcDNA3-EGFP Addgene #13031 Biological Samples Chicken embryos (Gallus gallus) AA Laboratories, Westminster, CA Quail embryos (Coturnix coturnix) McIntyre Poultry, Lakeside CA Chemicals, Peptides, and Recombinant Proteins Children’s Oncology Group Cell Culture and Xenograft Repository http://cogcell.org/ NRG1-EGF Domains NRG1α R&D Systems Cat#296-HR-050 BSA ThermoFisher Cat#BP-1600-100 NRG1β R&D Systems Cat#396-HB-050/CF Tryphostin AG 1478 Enzo Cat #ALX-270-036-M001 BPE BD Cat# 356123 Live Cell Imaging Solution Life Technologies Cat# A14291DJ Critical Commercial Assays Boyden Chamber Assay BD Immulon 4 ELISA well Dynatech Laboratories, Chantilly, VA microchips Caltech Microfluidic Foundry www.kni.caltech.edu/foundry Modified Zigmond Chamber Assay Walheim et al., 2012 Deposited Data Experimental Models: Cell Lines Experimental Models: Organisms/Strains Oligonucleotides Recombinant DNA Software and Algorithms Excel Microsoft https://products.office.com/en-us/excel SPSS IBM https://www.ibm.com/us-en/marketplace/spss-statistics ImageJ Manual Tracking ImageJ https://imagej.nih.gov/ij/plugins/track/track.html ImageJ Chemotaxis and Migration Tool ImageJ http://ibidi.com/software/chemotaxis_and_migration_tool AxioVision Rel.

Techniques: Virus, Recombinant, Cell Culture, Live Cell Imaging, Boyden Chamber Assay, Enzyme-linked Immunosorbent Assay, Modification, Software, Chemotaxis Assay, Migration

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