human recombinant nrg1β  (R&D Systems)

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a , Cartoon schematic of the HER2/HER4/NRG1β heterodimer depicts assembly of a ‘heart-shaped’ ectodomain dimer upon binding of a ligand/growth factor (GF) to HER4. Individual domains of the HER ectodomains are annotated as domain (D) I – IV. The intracellular kinase domains assemble into an asymmetric dimer in which HER2 adopts the receiver (activated) and HER4 the activator (inactive) positions, enforced by the interface mutations: HER2-V956R and HER4-I712Q, respectively. b-c , Structures of the near full-length HER2-V956R/HER4-I712Q complex (labeled HER2/HER4) bound to NRG1β or BTC. The ectodomain models are shown in cartoon representation fitted into the cryo-EM density. Only density for the ectodomain modules was observed. Domains I-IV are labeled DI-DIV. d , Overlay of HER2/HER4 heterodimers bound to NRG1β and BTC aligned on the HER2 chain (RMSD 0.835 Å). e , 3D classification analysis of HER2/HER4 heterodimers bound to NRG1β or BTC. Overlay of models in ribbon resulting from 3D classification of particles into four classes are shown (HER2/HER4/NRG1β 289,192 particles, HER2/HER4/BTC 148,541 particles). Models were aligned on the HER2 chain.

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Binding Assay, Labeling, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a , Overview of the HER2/HER4 purification strategy. HER2 features a G778D (GD) mutation to mediate Hsp90-independence. VR corresponds to HER2-V956R (receiver) and IQ to HER4-I712Q (activator). The mutant complex was used for all purification and structure determination steps shown in this figure and is referred to as HER2/HER4. b , Coomassie-stained SDS-PAGE gel analysis of the samples from the HER2/HER4 purification after ligand-mediated pulldown (1 st step) and MBP pulldown (2 nd step). c, Representative Size Exclusion Chromatography (SEC) profiles for liganded HER2/HER4 heterocomplexes. d , Representative 2D cryo-EM class averages of liganded HER2/HER4/NRG1β heterocomplexes. Box size is 321 Å. e , Western Blot showing that activation of HER2/HER4 heterodimers requires HER2 to adopt the kinase receiver function (HER2-VR) and HER4 to adopt the kinase activator (HER4-IQ) function in the heterodimer. Constructs were co-transfected into COS7 cells, starved overnight and stimulated with 10 nM ligand for 10 min at 37 °C. The blot is representative of three independent experiments.

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Purification, Mutagenesis, Staining, SDS Page, Size-exclusion Chromatography, Cryo-EM Sample Prep, Western Blot, Activation Assay, Construct, Transfection

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a, Cryo-EM map of HER2/HER4/NRG1β at different contour levels. b-c, GSFSC and 3DFSC plots of HER2/HER4/NRG1β. d, Local resolution map of HER2/HER4/NRG1β created using cryoSPARC 4. e, Cryo-EM map of HER2/HER4/BTC at different contour levels. f-g, GSFSC and 3DFSC plots of HER2/HER4/BTC. h, Local resolution map of HER2/HER4/BTC created using cryoSPARC 4.

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a, Overlays of indicated homo- and heterodimers. Heterodimer alignments were performed using the HER2 chain, alignments with HER4 homodimers were performed using the HER4 chain. The dotted line represents a C2 symmetry axis highlighting the asymmetry of heterodimers compared to near-perfect C2 symmetry observed for HER4/NRG1β homodimers. b , Individual receptors from the HER2-containing heterodimers were aligned using the HER2 chain or its co-receptor chain, as indicated. HER2-only is cryo-EM structure of the HER2 ECD with Pertuzumab and Trastuzumab Fab bound (PDB: 6OGE; Fabs not shown), HER2/HER3/NRG1β (PDB: 7MN5), HER2-S310F/HER3/NRG1β (PDB: 7MN6), HER2/EGFR/EGF (PDB: 8HGO). c , Comparison between indicated HER2 heterodimer structures. Structural models are overlayed on HER2 to highlight nuances with which HER2 engages its co-receptors. The same PDB codes were used as in b .

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Cryo-EM Sample Prep, Comparison

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a , cryo-EM density and model of the HER2/HER4/NRG1β domain II at two different orientations highlight two equally well resolved dimerization arms (DA). b , Hydrogen-bonds, cation-π interactions and salt bridges are depicted at the dimer interface, with other residues omitted for clarity. The HER2 and HER4 dimerization arms engage in the same set of polar interactions (insets A and B), except for a cation-π interaction between HER2 F279 with HER4 R306 (A) due to a substitution of the equivalent of HER3 R306 to L313 in HER2. Residues labelled “DI” are in receptor domain I while all others are in domain II (DII). Interface residues and hydrogen bonds were determined using UCSF ChimeraX. c , Known HER2 heterodimers are aligned using the HER2 chain to highlight positioning of the dimerization arms. d , Dimerization arm regions of selected HER receptor dimers are shown colored by B-factors. B-factor colors were scaled to represent max and min B-factor values within each structure corresponding to different absolute values across structures due to variability in their resolution. Distance measurements at fixed points highlight a correlation between asymmetrically distributed B-factors and asymmetrically engaged dimerization arms. e , Western Blot analysis of NR6 cell lysates transduced with indicated HER2 and HER4 constructs. Cells were starved for 4 h prior to stimulation with 10 nM NRG1β at 37° C for 10 min.

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Cryo-EM Sample Prep, Western Blot, Transduction, Construct

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a , Sequence alignment of HER receptor dimerization arm regions with conserved residues highlighted in red. Two aromatic residues that are known to engage in hydrogen bonding with the partner receptor are marked with (*). b , Full domains II ( DII ) for selected receptor dimers are shown in cartoon and all interface residues between two receptors within domains I and III ( DI-DIII ) are shown as sticks. Hydrogen bonds are indicated with dotted lines. Analysis was performed using UCSF ChimeraX. Domains IV are not resolved in most structures and are not included in this analysis. Canonical dimerization arm interactions involve domains DI and DII, while non-canonical interfaces, as seen for EGFR in the HER2/EGFR/EGF dimer and one EGFR/EREG monomer in the EGFR/EREG homodimer, engage DIII instead of DI. The following PDB codes were used: HER2/HER3/NRG1 β (PDB: 7MN5), HER2/EGFR/EGF (PDB: 8HGO), EGFR/EGF (PDB: 3NJP), HER4/NRG1β (PDB: 3U7U) and EGFR/EREG (PDB: 5WB7).

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a , Structures of full-length HER4 homodimers bound to either NRG1β or BTC. Only density for the ectodomain modules was observed in both structures, shown here as cartoon representation fitted into the cryo-EM density. b , Comparison between the NRG1β− and BTC-bound HER4 dimers. Angle measurements were derived using UCSF ChimeraX by defining an axis through each receptor in a dimer and measuring the angle between the two axes. c , Overlays of ribbon models obtained by 3D classification of particles into four distinct classes are shown for HER4 homodimers bound to NRG1β or BTC (205,726 particles HER4/NRG1 β and ∼274,540 particles HER4/BTC). Classification was performed in cryoSPARC using the heterogenous refinement with four identical start volumes and particles from final reconstructions are shown in (a) . d-e , Overlays of HER4 receptor homodimers bound to NRG1β or BTC show differences in the ligand binding pockets and how receptors assemble into dimers. Receptors were aligned as indicated in the panels. The HER4-NRG1β engages 3-4 salt bridges in the binding pocket, three of which are not present in HER4-BTC (shown in boxes). The salt bridge involving HER4 K35 can only be confidently observed in cryo-EM maps of one monomer (chain A).

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Cryo-EM Sample Prep, Comparison, Derivative Assay, Ligand Binding Assay, Binding Assay

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a , Overview of the HER4 purification strategy. Untagged, full-length HER4 was purified by growth factor (GF)-coated anti-FLAG resin. b , Coomassie-stained SDS-PAGE gel showing receptor samples after ligand-mediated receptor pulldown. c , SEC profiles of samples after ligand-mediated receptor pulldown using a Superose 6 increase 10/300 GL column. Elution fractions consistent with receptor dimers were used for negative-stain EM (NS-EM) and cryo-EM analyses. d , HER4/NRG1β NS-EM 2D class averages show receptor dimers with “heart”-shaped ectodomains and additional density for intracellular kinase domains. e , HER4/NRG1β and HER4/BTC cryo-EM 2D class averages show receptor dimers with “heart”-shaped extracellular domains without density for intracellular kinase domains. f , Cryo-EM volumes of HER4/NRG1β obtained from HER4/NRG1β preparations in an apo form, with afatinib, or with Mg 2+ AMPPNP bound. 10 mM afatinib was added to the culture medium during expression, 1 mM Mg 2+ AMPPNP was added prior to crosslinking with glutaraldehyde (after FLAG elution). Receptors were subjected to SEC and 1 mM Mg 2+ AMPPNP was again added prior to cryo-EM grid preparation. g , Overlay of models for volumes in f show all three volumes are identical.

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Purification, Staining, SDS Page, Cryo-EM Sample Prep, Expressing

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a, Cryo-EM map of HER4/NRG1β at different contour levels. b-c, GSFSC and 3DFSC plots of HER4/NRG1β. d, Local resolution map of HER4/NRG1β created using cryoSPARC4 e, Cryo-EM map of HER4/BTC at different contour levels. f, GSFSC and 3DFSC plots of HER4/BTC. g, Local resolution map of HER4/BTC created using cryoSPARC4.

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a , Overlay of three HER4/NRG1β ectodomain homodimers found in the asymmetric unit of the crystal structure (PDB: 3U7U) with the cryo-EM structure of full-length HER4/NRG1β. The crystal structure models are shown in grey. RMSDs for overlay of full dimers with the cryo-EM HER4/NRG1β dimer are 5.438 Å, 5.435 Å and 3.662 Å, respectively. b, HER4/NRG1β model built into C1 refined cryo-EM map was aligned across chains (chain A in one model aligned to chain B in another model). While the aligned chain showed a near perfect match (RMSD 1.42 Å), the other chain showed breaking of C2 symmetry. c , HER4/BTC model built into C1 refined cryo-EM map was aligned across chains (chain A in one model aligned to chain B in another model). While the aligned chain showed a near perfect match (RMSD 1.58 Å), the other chain showed breaking of C2 symmetry.

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a , Model of the HER4/NRG1β homodimer fitted into the cryo-EM density, lowpass-filtered to 6 Å, reveals multiple glycans that mediate intra- and interreceptor connections. Glycans are shown in blue. Insets A and B are close-up views of glycans connected to N138 and N253, and are shown at higher volume contour than the central heterodimer. Insets C and D are close-up views of glycans connected to N548, N576 and N358. D shows continuous glycan density originating from N576 of one receptor and connecting to N358 of the dimerization partner. Maps are shown at lower contour than in the central heterodimer. Various contour levels are shown in for reference. Arrows indicate regions in which the cryo-EM map from one glycan merges with density of glycans or polypeptide chains from different HER receptor sub-domains. b, Model of HER2/HER4/NRG1β fitted into cryo-EM density, lowpass-filtered to 6 Å, reveals intra-receptor glycosylation only. Insets A shows HER4 glycosylation on N548 and N576 pointing from HER4 domain IV to domain II, but less pronounced as observed in HER4 homodimers. Glycan connections between domain I and II in HER4, via N138 and N253-linked glycans, are comparable to the ones seen in HER4 homodimer shown in inset A . Inset B shows the equivalent glycan connections in domain I and II of HER2. Inset C reveals missing glycosylation sites at equivalent positions in HER2; G366, N556 and Q583 (pink).

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Cryo-EM Sample Prep, Glycoproteomics

Journal: bioRxiv

Article Title: Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

doi: 10.1101/2023.10.06.561161

Figure Lengend Snippet: a , Model of the HER4/NRG1β homodimer fitted into the cryo-EM density, lowpass-filtered to 6 Å, at various contour levels. Glycans are shown in blue. a , 3D classification of the HER4/NRG1β particles reveals strong continuous glycan density between two receptors within the dimer for class 3. c , Glycosylation site asparagines in EGFR, HER2, HER3 and HER4 are marked in blue, and shown in sphere representation. Glycosylation site asparagines involved in inter-receptor contacts in our HER4 structures, and the equivalent residues in other HER receptors are indicated by teal labels. d , Analysis of the cryo-EM map of the EGFR/EGF homodimer structure (PDB: 7SYD) at various contour levels suggests the presence of an inter-receptor glycan connection between N353 and N603, shown in blue.

Article Snippet: Cells were rinsed in PBS 5 hours post transfection, serum-starved for 16 hours and, if applicable, stimulated with 10 nM NRG1β (PeproTech) or BTC (PeproTech) for 10 minutes at 37° C. Cells were then washed with ice-cold PBS two times and lysed in 300 μl RIPA buffer (50 mM TRIS pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Roche Complete protease inhibitors, DNAse, 1 mM sodium orthovanadate, 1 mM sodium fluoride) on ice for 30 minutes.

Techniques: Cryo-EM Sample Prep, Glycoproteomics

Journal: Breast Cancer Research : BCR

Article Title: Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA

doi: 10.1186/s13058-021-01457-0

Figure Lengend Snippet: H1047R and E545K knockin cells are hyper-responsive to neuregulin-1 beta 1 (NRG1β) treatment. A Parental SK-BR-3 cells treated with NRG1β and lapatinib show rescue from lapatinib at high concentrations of NRG1β, but only partial rescue when treated with low doses of NRG1β (2 ng/mL). B The H1047R cells show complete rescue from lapatinib with all doses of NRG1β, even at the highest dose of lapatinib, suggesting that the mutant cells are more responsive to ligand stimulation than their wildtype counterparts. C The E545K cells also show complete rescue from lapatinib inhibition by all doses of NRG1β. Value shown are median cell counts relative to untreated controls. Error bars are ± standard deviation

Article Snippet: NRG1β EGF Domain (R&D Systems (Minneapolis, MN)) was added 24 h after cell plating, followed immediately by treatment with vehicle or lapatinib at four different concentrations (50, 100, 250, or 500 nM).

Techniques: Knock-In, Mutagenesis, Inhibition, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA

doi: 10.1186/s13058-021-01457-0

Figure Lengend Snippet: Addition of NRG1β de-sensitizes cells to lapatinib, co-treatment with AKT inhibitor or pertuzumab overcomes this effect. A Parental SKBR3 cells or PIK3CA mutant clones treated with lapatinib, AKT inhibitor GSK690693, or combination of the two drugs at a fixed molar ratio in the absence or presence of NRG1β for 72 h. Only H1047R knockin mutant cells show decreased sensitivity to lapatinib at baseline, consistent with results shown in Fig. . Treatment with NRG1β makes cells more resistant to lapatinib, and response is partially restored by co-treatment with GSK690693. The efficacy of the combination is greatest in the H1047R mutant cells as measured by the GR max response. Significant synergistic interactions are marked with an *. B Parental SKBR3 cells or PIK3CA mutant clones treated with lapatinib, pertuzumab, or combination of the two drugs at a fixed molar ratio in the absence or presence of NRG1β for 72 h. Combination of drugs resulted in synergistic growth inhibition at low doses in all three cell lines, but offered no benefit over lapatinib alone at higher doses. Addition of NRG1β-induced resistance to lapatinib in all three cell lines was abrogated with the addition of pertuzumab. Note that synergy could not be accurately calculated by the combination index method for these curves since it requires fitting dose-response curves and pertuzumab did not have any effect on the growth of cells as a monotherapy

Article Snippet: NRG1β EGF Domain (R&D Systems (Minneapolis, MN)) was added 24 h after cell plating, followed immediately by treatment with vehicle or lapatinib at four different concentrations (50, 100, 250, or 500 nM).

Techniques: Mutagenesis, Clone Assay, Knock-In, Inhibition

Journal: Science signaling

Article Title: Kinetics of receptor tyrosine kinase activation define ERK signaling dynamics *

doi: 10.1126/scisignal.aaz5267

Figure Lengend Snippet: (A) Endogenous ErbB4 in MCF7 cells was stimulated using saturating levels of NRG1β (25 nM/ 200 ng/ml) or NRG1α (1 μM/ 7 μg/ml). Activating phosphorylation of ErbB4 and ERK1/2, as well as total ErbB4 and ERK levels, were monitored by immunoblotting total cell lysates. Grb2 was used as a loading control. Blots are representative of at least three independent experiments. Statistical significance of differences was assessed using the Bonferroni-Dunn method (alpha = 0.05), with datasets for each point analyzed individually without assuming consistent SD.

Article Snippet: Recombinant human NRG1α (Cat. #296-HR) and NRG1β (Cat. #396-HB/CF) were obtained from R&D Systems.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: PLoS ONE

Article Title: Development and application of high-throughput screens for the discovery of compounds that disrupt ErbB4 signaling: Candidate cancer therapeutics

doi: 10.1371/journal.pone.0243901

Figure Lengend Snippet: (a) CEM/ErbB4 cells were manually stimulated with 10 nM NRG2β (full agonist positive control), 10 nM NRG2β/Q43L (partial agonist positive control), and mock (negative control). A single batch of lysate was assayed for ErbB4 tyrosine phosphorylation in eight independent trials using a manual sandwich ELISA. (b) CEM/ErbB4 cells were stimulated in six independent trials using a semi-automated protocol with 10 nM NRG1β (positive control) and mock (negative control). Each batch of lysate was assayed for ErbB4 tyrosine phosphorylation using a manual sandwich ELISA. (c) CEM/ErbB4 cells were stimulated using a semi-automated protocol with 10 nM NRG1β (positive control) and mock (negative control). A single batch of lysate was assayed for ErbB4 tyrosine phosphorylation in three independent trials using a semi-automated sandwich ELISA.

Article Snippet: Recombinant NRG1β was obtained from PeproTech (Catalog number 100–03, Lot number 0711316 and Lot number 0913316), and NRG2β isoforms and mutants have been described previously [ , , ].

Techniques: Positive Control, Negative Control, Phospho-proteomics, Sandwich ELISA

Journal: PLoS ONE

Article Title: Development and application of high-throughput screens for the discovery of compounds that disrupt ErbB4 signaling: Candidate cancer therapeutics

doi: 10.1371/journal.pone.0243901

Figure Lengend Snippet: (a) BaF3/EGFR+ErbB4 cells were stimulated with increasing concentrations of NRG1β in four independent trials using a semi-automated protocol. A semi-automated MTT assay was used to analyze cell proliferation 120 hours post-stimulation. Curves were fit to the data using GraphPad Prism to determine the EC 50 of NRG1β. The Z-factor for ErbB4-dependent cell proliferation stimulated by 0.3 nM NRG1β was calculated using the means and standard deviations of the positive (0.3 nM NRG1β) and negative (NRG1β-diluent) controls. (b) BaF3/EGFR+ErbB4 cells were treated with increasing concentrations of gefitinib in the presence of 0.3 nM NRG1β in four independent trials using a semi-automated protocol. A semi-automated MTT assay was used to analyze cell proliferation 120 hours post-stimulation. Curves were fit to the data using GraphPad Prism to determine the IC 50 of gefitinib against NRG1β.

Article Snippet: Recombinant NRG1β was obtained from PeproTech (Catalog number 100–03, Lot number 0711316 and Lot number 0913316), and NRG2β isoforms and mutants have been described previously [ , , ].

Techniques: MTT Assay

Journal: PLoS ONE

Article Title: Development and application of high-throughput screens for the discovery of compounds that disrupt ErbB4 signaling: Candidate cancer therapeutics

doi: 10.1371/journal.pone.0243901

Figure Lengend Snippet: (a-d) In three independent trials and using semi-automated processes, BaF3/EGFR+ErbB4 cells were stimulated with increasing concentrations of NRG1β in the presence or absence of gefitinib at 300 nM or candidates at 30 uM. A semi-automated MTT assay was used to analyze cell proliferation at 120 hours post-stimulation. Curves were fit to the data using GraphPad Prism to determine the EC 50 and E max of NRG1β in the presence and absence of gefitinib or the candidates. EC 50 and E max values are also shown in .

Article Snippet: Recombinant NRG1β was obtained from PeproTech (Catalog number 100–03, Lot number 0711316 and Lot number 0913316), and NRG2β isoforms and mutants have been described previously [ , , ].

Techniques: MTT Assay

Journal: PLoS ONE

Article Title: Development and application of high-throughput screens for the discovery of compounds that disrupt ErbB4 signaling: Candidate cancer therapeutics

doi: 10.1371/journal.pone.0243901

Figure Lengend Snippet: Effect of candidates on stimulation of cell proliferation by NRG1β.

Article Snippet: Recombinant NRG1β was obtained from PeproTech (Catalog number 100–03, Lot number 0711316 and Lot number 0913316), and NRG2β isoforms and mutants have been described previously [ , , ].

Techniques:

Journal: PLoS ONE

Article Title: Development and application of high-throughput screens for the discovery of compounds that disrupt ErbB4 signaling: Candidate cancer therapeutics

doi: 10.1371/journal.pone.0243901

Figure Lengend Snippet: (a-b) In three independent trials and using a modified version of our semi-automated processes, BaF3/EGFR+ErbB4 cells were treated with increasing concentrations of a candidate inhibitor or gefitinib (positive control inhibitor of NRG1β-induced proliferation) in the presence of 0.1 nM IL3 or 0.3 nM NRG1β. A semi-automated MTT assay was used to analyze cellular proliferation 120 hours post-stimulation. Curves were fit to the data using GraphPad Prism to determine the IC 50 value for each inhibitor against 0.1 nM IL3 and 0.3 nM NRG1β. IC 50 values are also shown in . *Extrapolated value.

Article Snippet: Recombinant NRG1β was obtained from PeproTech (Catalog number 100–03, Lot number 0711316 and Lot number 0913316), and NRG2β isoforms and mutants have been described previously [ , , ].

Techniques: Modification, Positive Control, MTT Assay

Journal: PLoS ONE

Article Title: Development and application of high-throughput screens for the discovery of compounds that disrupt ErbB4 signaling: Candidate cancer therapeutics

doi: 10.1371/journal.pone.0243901

Figure Lengend Snippet: Effect of increasing concentrations of candidate inhibitors on stimulation of cell proliferation by 0.3 nM NRG1β or 0.1 nM IL3.

Article Snippet: Recombinant NRG1β was obtained from PeproTech (Catalog number 100–03, Lot number 0711316 and Lot number 0913316), and NRG2β isoforms and mutants have been described previously [ , , ].

Techniques: Control

Journal: PLoS ONE

Article Title: Development and application of high-throughput screens for the discovery of compounds that disrupt ErbB4 signaling: Candidate cancer therapeutics

doi: 10.1371/journal.pone.0243901

Figure Lengend Snippet: (a-e) In three independent trials and using a modified version of our semi-automated processes, BaF3/EGFR+ErbB4 cells were treated with increasing concentrations of each candidate inhibitor in the presence of 0.1 nM IL3 or 0.3 nM NRG1β. A semi-automated MTT assay was used to analyze cellular proliferation 120 hours post-stimulation. Curves were fit to the data using GraphPad Prism to determine the IC 50 value for each candidate against 0.1 nM IL3 and 0.3 nM NRG1β. IC 50 values are also shown in .

Article Snippet: Recombinant NRG1β was obtained from PeproTech (Catalog number 100–03, Lot number 0711316 and Lot number 0913316), and NRG2β isoforms and mutants have been described previously [ , , ].

Techniques: Modification, MTT Assay

Journal: PLoS ONE

Article Title: Development and application of high-throughput screens for the discovery of compounds that disrupt ErbB4 signaling: Candidate cancer therapeutics

doi: 10.1371/journal.pone.0243901

Figure Lengend Snippet: (a-b) In three independent trials and using a modified version of our semi-automated processes, BaF3/EGFR+ErbB4 cells were treated with increasing concentrations of each candidate inhibitor in the presence of 0.1 nM IL3 or 0.3 nM NRG1β. A semi-automated MTT assay was used to analyze cellular proliferation 120 hours post-stimulation. Curves were fit to the data using GraphPad Prism to determine the IC 50 value for each candidate against 0.1 nM IL3 and 0.3 nM NRG1β.

Article Snippet: Recombinant NRG1β was obtained from PeproTech (Catalog number 100–03, Lot number 0711316 and Lot number 0913316), and NRG2β isoforms and mutants have been described previously [ , , ].

Techniques: Modification, MTT Assay