Journal: Cerebral Cortex (New York, NY)

Article Title: Temporal Dynam ics of the Neuregulin–ErbB Network in the Murine Prefrontal Cortex across the Lifespan

doi: 10.1093/cercor/bhz312

Figure Lengend Snippet: Details of reverse transcriptase quantitative PCR assays used in the current study

Article Snippet: Pan ErbB4 , Mm01256793_m1.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Sequencing

Journal: Cerebral Cortex (New York, NY)

Article Title: Temporal Dynam ics of the Neuregulin–ErbB Network in the Murine Prefrontal Cortex across the Lifespan

doi: 10.1093/cercor/bhz312

Figure Lengend Snippet: Gene expression profiles of v-erb homolog receptor tyrosine kinase genes ErbB2 (A), ErbB3 (B), and ErbB4 (C) in the postnatal mouse PFC from PND 1 through to 24 months of age. The geometric mean of Gapdh and βActin was used for normalization. Each data point represents an individual mouse PFC sample. N = 3–6 samples/age, n = 95 total lifespan. *P < 0.05, **P < 0.01, ***P < 0.001 significant correlation with age within lifestage (early postnatal n = 25, prepubertal n = 21, postpubertal n = 14, adulthood through aging n = 25). ###P < 0.001 significant correlation across postnatal age. Lines represent best fit expression trajectory within lifestage (gray) or across entire postnatal period (black). M, month.

Article Snippet: Pan ErbB4 , Mm01256793_m1.

Techniques: Gene Expression, Expressing

Journal: Cerebral Cortex (New York, NY)

Article Title: Temporal Dynam ics of the Neuregulin–ErbB Network in the Murine Prefrontal Cortex across the Lifespan

doi: 10.1093/cercor/bhz312

Figure Lengend Snippet: Gene expression profiles of ErbB4 splice isoforms JMa (A), JMb (B) CYT1 (C), and CYT2 (D) as well as the long noncoding RNA Miat (E) in the postnatal mouse PFC from PND 1 through to 24 months of age. The geometric mean of Gapdh and βActin was used for normalization. Correlation analysis of normalized Miat expression to the ratio of JMa:JMb (white data points) and CYT1:CYT2 (black data points) ErbB4 transcripts (F). Each data point represents an individual mouse PFC sample. N = 3–6 samples/age, n = 95 total lifespan. *P < 0.05, **P < 0.01, ***P < 0.001 significant correlation with age within lifestage (early postnatal n = 25, prepubertal n = 21, postpubertal n = 14, adulthood through aging n = 25). ###P < 0.001 significant correlation across postnatal age. Lines represent best fit expression trajectory within lifestage (gray) or across entire postnatal period (black). M, month.

Article Snippet: Pan ErbB4 , Mm01256793_m1.

Techniques: Gene Expression, Expressing

Journal: Cerebral Cortex (New York, NY)

Article Title: Temporal Dynam ics of the Neuregulin–ErbB Network in the Murine Prefrontal Cortex across the Lifespan

doi: 10.1093/cercor/bhz312

Figure Lengend Snippet: Expression profiles of Nrg–ErbB proteins across murine cortical development and aging. Densitometry analysis of western blots for full-length protein products of Neuregulin 1 (A), Neuregulin 3 (B), and ErbB4 (C) in the postnatal mouse PFC at PND 1, PND 35, 3 months (M), and 24 M. Expression of β-Actin was used for normalization. Data represent mean ± SEM. n = 3 samples/age.

Article Snippet: Pan ErbB4 , Mm01256793_m1.

Techniques: Expressing, Western Blot

Journal: Cerebral Cortex (New York, NY)

Article Title: Temporal Dynam ics of the Neuregulin–ErbB Network in the Murine Prefrontal Cortex across the Lifespan

doi: 10.1093/cercor/bhz312

Figure Lengend Snippet: Summary of cortical expression trajectories of the Nrg-ErbB network across the murine lifespan

Article Snippet: Pan ErbB4 , Mm01256793_m1.

Techniques: Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms

doi: 10.1186/s12964-019-0388-5

Figure Lengend Snippet: ErbB4 is commonly expressed in human MPNSTs and MPNST-derived cell lines. a - f Representative images of erbB4 immunostaining of FFPE sections of human MPNSTs demonstrates different grades of erbB4 staining compared to isotype matched negative control b . The erbB4 grading is represented numerically on a scale between 1+ to 4+. A subset of erbB4 positive tumor samples displayed prominent nuclear staining ( c - f ) and others displayed exclusive non-nuclear membranous staining ( a ). Red channel (Alexa 568, erbB4). Blue channel (Hoechst, nuclei). Scale bar represents 100um. g Real time PCR analysis showing relative mRNA expression of erbB4 mRNA using non-isoform discriminatory PCR primers; mRNA levels are shown relative to human Schwann cells. GAPDH mRNA was used as the loading control for normalization. h ErbB4 protein expression of by immunoblot analysis of whole cell lysates derived from human MPNST derived cell lines. GAPDH was used as a loading control

Article Snippet: The TaqMan primer sets used were: pan-erbB4: Hs00171783_m1 (spans exons 12–13, detects all isoforms), Hs00955522_m1 (spans exons 26–27; CYT1 specific), Hs00955509_m1 (spans exons 14–15; JMb specific), Hs00955511_m1 (spans exons 16–17; JMa specific), a custom set for CYT2 (spans exons 25–27; 5′ primer: 5′-CAACATCCCACCTCCCATCTATAC-3′, 3′ primer: 5′ACACTCCTTGTTCAGCAGCAAA-3′, probe a: 5′AATTGACTCGAATAGGAACCAGTTTGTATACCGAGAT-3′) and GAPDH (Hs99999905_m1, Mm99999915_g1).

Techniques: Derivative Assay, Immunostaining, Staining, Negative Control, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms

doi: 10.1186/s12964-019-0388-5

Figure Lengend Snippet: Loss of ErbB4 in human and mouse MPNST cells inhibits proliferation and survival. a Compared to non-targeting controls, erbB4 knockdown in cells had a decrease in cellular proliferation. b Immunoblots demonstrating reduced erbB4 expression in low erbB4 expressing cells (T265-2c, S462) and high expressing cells (MPNST642) cells infected with erbB4 shRNAs relative to cells infected with a non-targeting control. **, p -value≤0.01; ***, p -value≤0.001; ****, p -value≤0.0001. c Representative erbB4 immunostaining of P 0 -GGFβ3; Trp53 +/− ; Erbb4 flox/flox GEM mouse tumors showing erbB4 positivity compared to isotype matched negative controls ( h ). These representative erbB4 immunostains also show punctate membranous staining, with immunoreactivity present in almost all tumor cells (4+ staining). d ) Kaplan-Meier survival curve of /flox mice shows that these animals survive an average of 210 days, similar to the parent line P 0 -GGFβ3; Trp53 +/− mice. e ) Like their parent line, P 0 -GGFβ3; Trp53 +/− ; Erbb4 flox/flox mice develop hypercellular neoplasms comprised of atypical spindled cells with numerous mitotic figures. As in human MPNSTs, these tumors are also immunoreactive for both S100β ( f ) and nestin ( g ). h Isotype-matched negative control for erbB4 staining of mouse tumors. i Representative infection efficiency of UBI MPNST cell lines. MPNSTs with intact Erbb4 alleles (control, non-recombined) have a spindled morphology similar to that of wild-type Schwann cells, whereas knockout cells (Cre) are more ameboid. Nuclear blebbing was evident in a subset of Erbb4- ablated tumor cells (arrow). Images were imaged at 40X on Brightfield and GFP channels. Infected cells are represented on the green channel due to GFP target sequence in the adenovirus. j , k Decreased cellular viability ( j ) and proliferation ( k ) was observed in the Erbb4 knockout cells (Cre) compared to the control cells from three independent tumor cultures. *, p -value≤0.05; **** p -value≤0.0001 for comparisons of recombined ( Erbb4 -) with non-recombined ( Erbb4+) alleles. Red channel (Alexa 565, erbB4). Blue channel (Hoechst, nuclei)

Article Snippet: The TaqMan primer sets used were: pan-erbB4: Hs00171783_m1 (spans exons 12–13, detects all isoforms), Hs00955522_m1 (spans exons 26–27; CYT1 specific), Hs00955509_m1 (spans exons 14–15; JMb specific), Hs00955511_m1 (spans exons 16–17; JMa specific), a custom set for CYT2 (spans exons 25–27; 5′ primer: 5′-CAACATCCCACCTCCCATCTATAC-3′, 3′ primer: 5′ACACTCCTTGTTCAGCAGCAAA-3′, probe a: 5′AATTGACTCGAATAGGAACCAGTTTGTATACCGAGAT-3′) and GAPDH (Hs99999905_m1, Mm99999915_g1).

Techniques: Knockdown, Western Blot, Expressing, Infection, Control, Immunostaining, Staining, Negative Control, Knock-Out, Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms

doi: 10.1186/s12964-019-0388-5

Figure Lengend Snippet: Erbb4 ablation inhibits allograft growth and alters tumor histopathology in Nude mice. a and b ) Erbb4- null cells from three separate tumor lines show reduced graft volume ( a ) and mass ( b ) compared to erbB4 expressing controls. p -values ≤0.05 are designated with an asterisk (*), Tumor mass p -values are as follows: UBI-1: 0.049, UBI-2: 0.069, and UBI-3: 0.005; F-Test. c Representative histologic images of a UBI GFP control tumor and a UBI Cre tumor taken at 40X. Arrows in Ctl indicate the multinucleated cells that are commonly seen in these tumors. Note that these multinucleated cells are virtually absent in the Erbb4 -null grafts. d Representative chromogenic erbB4 stained images of UBI GFP control tumor and UBI Cre tumors displaying decreased erbB4 expression in Cre ablated xenografts compared to control tumors taken at 20X

Article Snippet: The TaqMan primer sets used were: pan-erbB4: Hs00171783_m1 (spans exons 12–13, detects all isoforms), Hs00955522_m1 (spans exons 26–27; CYT1 specific), Hs00955509_m1 (spans exons 14–15; JMb specific), Hs00955511_m1 (spans exons 16–17; JMa specific), a custom set for CYT2 (spans exons 25–27; 5′ primer: 5′-CAACATCCCACCTCCCATCTATAC-3′, 3′ primer: 5′ACACTCCTTGTTCAGCAGCAAA-3′, probe a: 5′AATTGACTCGAATAGGAACCAGTTTGTATACCGAGAT-3′) and GAPDH (Hs99999905_m1, Mm99999915_g1).

Techniques: Histopathology, Expressing, Control, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms

doi: 10.1186/s12964-019-0388-5

Figure Lengend Snippet: a Quantification of Ki67 labeling indices in three allografted tumor lines. b Quantification of TUNEL labeling indices in the same tumor lines. ****, p -value≤0.0001 for comparisons of recombined Erbb4 allele (erbB4 -) grafts to grafts with non-recombined Erbb4 alleles (erbB4+). c - i Representative CD31 staining of control erbB4 positive tumors ( c , e ) and erbB4 negative tumors ( g , i ) with Bisbenzimide counterstain ( b , h ) for cell nuclei detection. A non-immune isotype matched primary was used as a negative control ( f ). Images were acquired at 40X. Scale bars represent 100 um. j Quantification of representative immunofluorescent images using FIJI. *, p -value≤0.05; **** p -value≤0.0001 for comparisons of recombined ( Erbb4 -) with non-recombined ( Erbb4+) alleles

Article Snippet: The TaqMan primer sets used were: pan-erbB4: Hs00171783_m1 (spans exons 12–13, detects all isoforms), Hs00955522_m1 (spans exons 26–27; CYT1 specific), Hs00955509_m1 (spans exons 14–15; JMb specific), Hs00955511_m1 (spans exons 16–17; JMa specific), a custom set for CYT2 (spans exons 25–27; 5′ primer: 5′-CAACATCCCACCTCCCATCTATAC-3′, 3′ primer: 5′ACACTCCTTGTTCAGCAGCAAA-3′, probe a: 5′AATTGACTCGAATAGGAACCAGTTTGTATACCGAGAT-3′) and GAPDH (Hs99999905_m1, Mm99999915_g1).

Techniques: Labeling, TUNEL Assay, Staining, Control, Negative Control

Journal: Cell Communication and Signaling : CCS

Article Title: ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms

doi: 10.1186/s12964-019-0388-5

Figure Lengend Snippet: Erbb4 is not essential for Ras hyperactivation. a A comparison of Ras activation in Erbb4 -null (Cre) and Erbb4 -expressing control (Ctl) UBI 1–3 MPNST cells shows that Erbb4 loss does not affect Ras activation. b Immunocytochemistry staining of Erbb4 -null (Cre) and Erbb4 -expressing control (Ctl) UBI MPNSTs cells displays reduced erbB4 expression in adeno-viral infected cells (GFP positive). Representative erbB4 staining of control erbB4 positive tumors (Ctl) and erbB4 negative tumors (Cre) with Bisbenzimide counterstain for cell nuclei detection. A non-immune isotype matched primary was used as a negative control. Images were acquired at 20X. Scale bars represent 100 um

Article Snippet: The TaqMan primer sets used were: pan-erbB4: Hs00171783_m1 (spans exons 12–13, detects all isoforms), Hs00955522_m1 (spans exons 26–27; CYT1 specific), Hs00955509_m1 (spans exons 14–15; JMb specific), Hs00955511_m1 (spans exons 16–17; JMa specific), a custom set for CYT2 (spans exons 25–27; 5′ primer: 5′-CAACATCCCACCTCCCATCTATAC-3′, 3′ primer: 5′ACACTCCTTGTTCAGCAGCAAA-3′, probe a: 5′AATTGACTCGAATAGGAACCAGTTTGTATACCGAGAT-3′) and GAPDH (Hs99999905_m1, Mm99999915_g1).

Techniques: Comparison, Activation Assay, Expressing, Control, Immunocytochemistry, Staining, Infection, Negative Control

Journal: Cell Communication and Signaling : CCS

Article Title: ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms

doi: 10.1186/s12964-019-0388-5

Figure Lengend Snippet: RNA-Seq pathway analysis of differentially affected genes in erbB4 knockout UBI MPNST cells. Partek analysis of NextGen RNA sequencing identified differentially expressed genes. Genes with a fold change of at least 1.5 up or down were put through Panther gene over representation enrichment analysis to identify erbB4 affected signaling pathways ( a ) and molecular function activity ( b ). Graphs represent the ratio of over-represented genes differentially affected by erbB4 loss compared to expected reference representation. This analysis highlights the role of erbB4 in migration, angiogenesis and PLCγ signaling

Article Snippet: The TaqMan primer sets used were: pan-erbB4: Hs00171783_m1 (spans exons 12–13, detects all isoforms), Hs00955522_m1 (spans exons 26–27; CYT1 specific), Hs00955509_m1 (spans exons 14–15; JMb specific), Hs00955511_m1 (spans exons 16–17; JMa specific), a custom set for CYT2 (spans exons 25–27; 5′ primer: 5′-CAACATCCCACCTCCCATCTATAC-3′, 3′ primer: 5′ACACTCCTTGTTCAGCAGCAAA-3′, probe a: 5′AATTGACTCGAATAGGAACCAGTTTGTATACCGAGAT-3′) and GAPDH (Hs99999905_m1, Mm99999915_g1).

Techniques: RNA Sequencing, Knock-Out, Protein-Protein interactions, Activity Assay, Migration

Journal: Cell Communication and Signaling : CCS

Article Title: ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms

doi: 10.1186/s12964-019-0388-5

Figure Lengend Snippet: Erbb4 increases the phosphorylation of a number of other cytoplasmic kinases, independent of Ras activation. a Graphical representation of quantified kinase arrays comparing the levels of baseline phosphorylation to NRG1 stimulated phosphorylation in Erbb4 -expressing UBI MPNST cells. The graph includes the subset of kinases whose phosphorylation was altered following NRG1β stimulation. b Graphical representation of quantified kinase arrays comparing the levels of baseline phosphorylation of control Erbb4- intact cells to NRG1 stimulated ErbB4 -intact and Erbb4-null UBI MPNST cells to determine NRG1 dependent and ErbB4 dependent kinases. c ) Quantification of a subset of the non-responsive kinases ErbB4 -intact compared to ErbB4 -ablated to identify targets positively regulated by ErbB4 independent of stimulation. d ) Quantification of a subset of the non-responsive kinases ErbB4 -intact compared to ErbB4 -ablated to identify compensatory targets resulting from ErbB4 -ablation independent of stimulation. Quantification of the kinases differentially phosphorylated was quantified per the manufacturer’s protocol using ImageJ

Article Snippet: The TaqMan primer sets used were: pan-erbB4: Hs00171783_m1 (spans exons 12–13, detects all isoforms), Hs00955522_m1 (spans exons 26–27; CYT1 specific), Hs00955509_m1 (spans exons 14–15; JMb specific), Hs00955511_m1 (spans exons 16–17; JMa specific), a custom set for CYT2 (spans exons 25–27; 5′ primer: 5′-CAACATCCCACCTCCCATCTATAC-3′, 3′ primer: 5′ACACTCCTTGTTCAGCAGCAAA-3′, probe a: 5′AATTGACTCGAATAGGAACCAGTTTGTATACCGAGAT-3′) and GAPDH (Hs99999905_m1, Mm99999915_g1).

Techniques: Phospho-proteomics, Activation Assay, Expressing, Control

Journal: Cell Communication and Signaling : CCS

Article Title: ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms

doi: 10.1186/s12964-019-0388-5

Figure Lengend Snippet: MPNSTs are sensitive to inhibition of specific erbB4 activated signaling pathways. a - c Cell viability was assessed in three log phase MPNST cell lines in the presence of designated inhibitor. Values represent cell viability after 72 h of drug treatment normalized to 0 h. After normalization, cell viability for each drug dose was compared to vehicle control. PLC-γ inhibition effects cell viability in a dose dependent manner a . STAT3 inhibition significantly affects cell viability ( b ). STAT5 inhibition modestly affects cell viability ( c )

Article Snippet: The TaqMan primer sets used were: pan-erbB4: Hs00171783_m1 (spans exons 12–13, detects all isoforms), Hs00955522_m1 (spans exons 26–27; CYT1 specific), Hs00955509_m1 (spans exons 14–15; JMb specific), Hs00955511_m1 (spans exons 16–17; JMa specific), a custom set for CYT2 (spans exons 25–27; 5′ primer: 5′-CAACATCCCACCTCCCATCTATAC-3′, 3′ primer: 5′ACACTCCTTGTTCAGCAGCAAA-3′, probe a: 5′AATTGACTCGAATAGGAACCAGTTTGTATACCGAGAT-3′) and GAPDH (Hs99999905_m1, Mm99999915_g1).

Techniques: Inhibition, Protein-Protein interactions, Control

Journal: Cell Communication and Signaling : CCS

Article Title: ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms

doi: 10.1186/s12964-019-0388-5

Figure Lengend Snippet: MPNSTs are sensitive to knockdown of specific erbB4 activated signaling molecules. a - c ) Cell proliferation was assessed in three log phase MPNST cell lines in the presence of multiple designated PLCγ( a ), STAT3 ( b ), or STAT5 ( c ) shRNA’s or non-targeting controls. Values represent cell number over 5 days of shRNA knockdown normalized to 0 h. PLC-γ inhibition effects cell proliferation in a cell-type dependent manner ( a ). STAT3 inhibition significantly affects cell proliferation ( b ). STAT5 inhibition significantly affects cell proliferation ( c )

Article Snippet: The TaqMan primer sets used were: pan-erbB4: Hs00171783_m1 (spans exons 12–13, detects all isoforms), Hs00955522_m1 (spans exons 26–27; CYT1 specific), Hs00955509_m1 (spans exons 14–15; JMb specific), Hs00955511_m1 (spans exons 16–17; JMa specific), a custom set for CYT2 (spans exons 25–27; 5′ primer: 5′-CAACATCCCACCTCCCATCTATAC-3′, 3′ primer: 5′ACACTCCTTGTTCAGCAGCAAA-3′, probe a: 5′AATTGACTCGAATAGGAACCAGTTTGTATACCGAGAT-3′) and GAPDH (Hs99999905_m1, Mm99999915_g1).

Techniques: Knockdown, shRNA, Inhibition

Journal: Cell Communication and Signaling : CCS

Article Title: ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms

doi: 10.1186/s12964-019-0388-5

Figure Lengend Snippet: Schematic highlighting the signaling cascades that are dependent on erbB4 in MPNSTs. The STAT3/5 and PI3K pathway is positively regulated by erbB4 in a NRG1β-dependent manner. Kinases validated experimentally are demarcated with an asterisk

Article Snippet: The TaqMan primer sets used were: pan-erbB4: Hs00171783_m1 (spans exons 12–13, detects all isoforms), Hs00955522_m1 (spans exons 26–27; CYT1 specific), Hs00955509_m1 (spans exons 14–15; JMb specific), Hs00955511_m1 (spans exons 16–17; JMa specific), a custom set for CYT2 (spans exons 25–27; 5′ primer: 5′-CAACATCCCACCTCCCATCTATAC-3′, 3′ primer: 5′ACACTCCTTGTTCAGCAGCAAA-3′, probe a: 5′AATTGACTCGAATAGGAACCAGTTTGTATACCGAGAT-3′) and GAPDH (Hs99999905_m1, Mm99999915_g1).

Techniques: