ligand for egfr  (R&D Systems)

Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A) Hematoxylin and eosin (H&E) staining of normal duodenum containing Brunner’s glands (nDUO-BG) and duodenal neuroendocrine tumor (DNET). Dashed boxes indicate regions shown at higher magnification. (B) Immunohistochemical staining for synaptophysin (SYP) confirming neuroendocrine differentiation in DNET. (C-D) Immunohistochemical staining for TGFα and EREG in tumor-associated Brunner’s glands (tBG) and DNET. Dashed boxes indicate tumor-gland interfaces. (E-F) Quantification of TGFα and EREG expression by H-score in nDUO-BG, tBG, and DNET. Data are mean ± SEM; ns, not significant; ****P < 0.0001. (G) EGFR immunostaining in nDUO-BG and DNET showing heterogeneous expression across tissues. (H) Menin immunostaining in nDUO-BG and DNET. (I) Representative FFPE DNET specimens showing cytoplasmic or near-absent Menin expression, accompanied by strong TGFα and EREG staining within tumor cells. (J) Quantification of Menin nuclear-to-cytoplasmic (N/C) ratio in nDUO-BG and DNET. Data are mean ± SEM; ****P < 0.0001. Images were taken at 100X, 200X and 400X. Scale bars: 100 μm (low magnification) and 50 μm (high magnification).

Article Snippet: After 24 h, cells were serum-starved for 24 h, followed by treatment with either recombinant human eregulin (EREG) protein (10 nM), a potent ligand for EGFR (R&D systems, #1195-EP) or 10 μM Forskolin (FSK, ThermoFisher, #66575-29-9), activator of adenylyl cyclase and cyclic AMP or and 10nM phorbol 12-myristate 13-acetate (TPA; Sigma-Aldrich, #P8139) for 4-8 h. Cells were lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, #E1980) according to the manufacturer’s instructions.

Techniques: Staining, Immunohistochemical staining, Expressing, Immunostaining

Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A) Multiple sequence alignment of the Menin C-terminal region from the indicated vertebrate species showing strong conservation of a basic residue–rich motif encompassing Ser487. Conserved basic residues and Ser487 are highlighted. (B) Schematic of human Menin illustrating the position of Ser487 within NLS1. The expanded sequence highlights Ser487 and surrounding basic residues; constructs used in this study. (C) Immunoblot analysis of AGS cells expressing FLAG-tagged wild-type Menin or Ser487 mutants (S487A, S487D) following treatment with EREG, FSK, or TPA. Whole-cell lysates were probed with antibodies against phospho-Ser487 Menin, FLAG-Menin, and GAPDH. (D, E) Immunoblot analysis of MKN-45G and KATO III cells expressing wild-type Menin following stimulation with EREG, FSK, or TPA. Blots were probed for phospho-Ser487 Menin, FLAG-Menin, and β-tubulin. (F, H) Quantification of phospho-Ser487 Menin in AGS, KATO III and MKN-45G cells. (I) Immunoblot analysis of AGS cells examining activation of cAMP and EGFR downstream kinases under the indicated conditions. (J) Densitometric quantification of signalling outputs shown in (I), expressed as fold change relative to vector control. (K) Time-course of Ser487 phosphorylation in AGS cells stimulated with TPA in the presence of kinase inhibitors; MEK inhibitor (U0126), AKT inhibitor (MK-2206), PKC inhibitor (Gö6983), or combined MEK+AKT inhibition. (L) Quantification of Ser487 phosphorylation kinetics following TPA stimulation with the indicated inhibitors. (M) Area-under-the-curve (AUC) analysis of phosphorylation in (L). Data are presented as mean ± SEM; individual data points represent independent biological replicates (n = 3). Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test (*P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant).

Article Snippet: After 24 h, cells were serum-starved for 24 h, followed by treatment with either recombinant human eregulin (EREG) protein (10 nM), a potent ligand for EGFR (R&D systems, #1195-EP) or 10 μM Forskolin (FSK, ThermoFisher, #66575-29-9), activator of adenylyl cyclase and cyclic AMP or and 10nM phorbol 12-myristate 13-acetate (TPA; Sigma-Aldrich, #P8139) for 4-8 h. Cells were lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, #E1980) according to the manufacturer’s instructions.

Techniques: Sequencing, Residue, Construct, Western Blot, Expressing, Activation Assay, Plasmid Preparation, Control, Phospho-proteomics, Inhibition, Comparison

Journal: bioRxiv

Article Title: Mapping mesenchymal diversity in the developing human intestine and organoids

doi: 10.1101/2025.07.22.665939

Figure Lengend Snippet: UMAP projections of the major cell class composition in a Xenium image of both an A) EGF-tHIO and B) EREG-tHIO. C) Stacked bar graph summarizing the percent cellular composition of the EGF-tHIO (left) and EREG-tHIO (right) samples. Cell ID masks generated in XE depicting 1) the entire sample acquired for the D) EGF-tHIO and E) EREG-tHIO. Scale bar = 5000µm. The white box indicates the ROI depicted in D2/E2-D7/E7. D2-7 and E2-7 display the cell sub-type distribution of each major cell class identified using the Xenium panel visualized as 2) the major cell class mask, 3) the epithelium sub-types, 4) the fibroblast sub-types, 5) the SMC-related sub-types, 6) the ENS sub-types, and 7) the endothelium sub-types. Scale bar = 500µm Cell classes are color matched in A-E ; epithelium (gold), fibroblasts (navy), muscularis mucosa (MM, light green), SMC and pericytes (dark green), ENS (yellow), and endothelium (cyan). No immune cells were observed in either tHIO sample. DAPI staining of cell nuclei depicted in grey.

Article Snippet: Organoid basal growth media was supplemented with epidermal growth factor (EGF) (100 ng/mL R&D Systems Cat#236-EG-01M) or Epiregulin (EREG) (10 ng/mL R&D Systems Cat#1195-EP-025/CF) with Noggin-Fc (100ng/mL, purified from conditioned media), and R-Spondin1 (5% conditioned medium) for the first three days of culture to pattern a proximal small intestine.

Techniques: Generated, Staining

Journal: bioRxiv

Article Title: Mapping mesenchymal diversity in the developing human intestine and organoids

doi: 10.1101/2025.07.22.665939

Figure Lengend Snippet: UMAP projections of the fibroblast cell class composition in a Xenium image of both an A) EGF-tHIO and B) EREG-tHIO. C) Stacked bar graph summarizing the percent cellular composition of the EGF-tHIO (left) and EREG-tHIO (right) samples. Cell ID masks generated in XE depicting D) EGF-tHIO and E) EREG-tHIO samples. 1) XE cell mask summarizing the major cell classes for the tHIOs to landmark fibroblast population locations. Fibroblast sub-type distribution is depicted as 2) the F3 + SECs, 3) the FABP5 + early LPFs, 4) the ADAMDEC1 + late LPFs, 5) the SHISA3 + SMFs, and 6) the CXCL13 + Fibroblasts. 7) depicts the composite mask summarizing all fibroblast cell types in the tHIOs. Scale bars = 100µm

Article Snippet: Organoid basal growth media was supplemented with epidermal growth factor (EGF) (100 ng/mL R&D Systems Cat#236-EG-01M) or Epiregulin (EREG) (10 ng/mL R&D Systems Cat#1195-EP-025/CF) with Noggin-Fc (100ng/mL, purified from conditioned media), and R-Spondin1 (5% conditioned medium) for the first three days of culture to pattern a proximal small intestine.

Techniques: Generated

Journal: Cell stem cell

Article Title: Coordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature.

doi: 10.1016/j.stem.2025.02.007

Figure Lengend Snippet: Figure 1. EREG-HIOs grown in vitro spontaneously and simultaneously pattern endothelium, smooth muscle, and neural components (A) Schematic of HIO-directed differentiation using standard EGF conditions (gray) and experimental EREG conditions (pink). (B) UMAP visualization of snRNA-seq from 28-day in vitro-grown EREG-HIOs in 10 ng/mL of EREG (n = 1 sequencing run of over 20 combined HIOs). (C) Dot plot visualization for expression of canonical markers of neurons (S100B, PLP1, STMN2, and ELAVL4), endothelial cells (CDH5, KDR, ECSCR, and CLDN5), mesenchyme (COL1A1, COL1A2, and DCN), smooth muscle (ACTA2, TAGLN, ACTG2, and MYLK), epithelium (EPCAM, CDH1, CDX2, and CLDN4), immune cells (PTPRC, ARHGDIB, and CORO1A), and proliferative cells (MKI67 and TOP2A) in EREG-grown (10 ng/mL) HIOs.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Epidermal Growth Factor (EGF) R&D Systems Cat#236-EG Epiregulin (EREG) R&D Systems Cat#1195-EP Recombinant Human Noggin-FC Purified from HEK293 cells expressing FC-tagged Noggin N/A Human R-Spondin1 Purified from Conditioned Medium from Cultrex HA-R-Spondin1-FC 293 T-Cells Cat# 3710-001-01 B27 Supplement Thermo Fisher Cat#17504044 HEPES Gibco Cat#15630080 GlutaMAX Gibco Cat#35050061 Penicillin-Streptomycin Thermo Fisher Cat#15140122 mTeSR 1 Stemcell Technologies Cat#85850 RPMI 1640 Thermo Fisher Cat#11875093 FBS Sigma Cat#12103C Dimethyl phenyl piperazinium Sigma Cat#D5891 NG-nitro-L-arginine methyl ester Sigma Cat#N5751 Atropine sulfate salt monohydrate Sigma Cat#A0132 Neurotoxin tetrodotoxin Tocris Cat#1078 M199 Gibco Cat#11150067 Heparin Sigma Cat#H3149-100KU FGF2 R&D Systems Cat#236-EG-01M N-acetylcysteine Sigma Cat#A9165-25G Accutase Corning Cat#MT2508CI Carbamyl-b-methylcholine chloride Sigma Cat#C5259 Scopolamine hydrobromide Tocris Cat#1414 Fibrinogen from bovine plasma Sigma Cat#F8630 Human Fibrinogen 1 Plasminogen Depleted Enzyme Research Lab Cat#FIB-1 X-Vivo 20 Lonza Cat#190995 Thrombin from bovine plasma Sigma Cat#T4648 StemSpan SFEM Stemcell Technologies Cat#9650 Knockout Serum Gibco Cat#10828010 Heparin Sigma Cat#H3149 FGF2 R&D Systems Cat#233-FB-MTO Aprotinin Sigma Cat#A6106 Critical commercial assays Neural Tissue Dissociation Kit Miltenyi Cat#130-092-628 103 Genomics Chromium Nuclei Isolation Kit 103 Genomics Cat#1000493 103 Chromium Controller v3 chemistry 103 Genomics Cat# 1000268 MagMax-96 Total RNA Isolation Kit/machine Thermo Fisher Cat#AM1830 SuperScript VILO cDNA Kit Thermo Fisher Cat#11754250 QuantiTect SYBR Green PCR Kit QIAGEN Cat#204145 MycoAlert Mycoplasma Detection Kit Lonza Cat#LT07-318 Proteome Profiler Human Phospho-Kinase Array Kit R&D Systems Cat#ARY003C Deposited data Raw scRNAseq data (human fetal intestine) Holloway et al.24 ArrayExpress: E-MTAB-9489 Raw scRNAseq data (HIO whole cell) This study ArrayExpress: E-MTAB-13463 Raw snRNAseq data (HIO and tHIO nuclei) This study ArrayExpress: E-MTAB-13469 Experimental models: Cell lines H9 ESC WiCell NIH registry#0062, RRID: CVCL_9773, female iPSC WTC11 Coriell Institute RRID: CVCL_Y803, male (Continued on next page) e2 Cell Stem Cell 32, 1–12.e1–e9, April 3, 2025

Techniques: In Vitro, Sequencing, Expressing

Journal: Cell stem cell

Article Title: Coordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature.

doi: 10.1016/j.stem.2025.02.007

Figure Lengend Snippet: Figure 2. EREG-grown HIOs further mature and spatially organize after transplantation into murine kidney capsule (A) Schematic timeline of HIO transplantation experiment. (B) Representative IF staining of human fetal intestine (left; 127 days post conception), EGF-grown tHIO (middle; 12 weeks), and EREG-grown tHIO (right; 12 weeks) stained for the presence of smooth muscle (SM22; green), epithelium (ECAD; blue), and neurons (TUBB3; pink) in top panels. Bottom panels show stains for the presence of smooth muscle (SM22; green), epithelium (ECAD; blue), and endothelial cells (PECAM; pink). All scale bars, 50 mm. (C) Quantification of neurons (TUBB3+ cells) and human endothelial cell (PECAM+ cells) stains normalized to total area of section (DAPI+) for a 127-day post conception developing human intestine, EREG-grown, and EGF-grown tHIOs (n = 6 transplanted organoids and sections of human intestine). Statistical sig- nificance was determined using an ordinary one-way ANOVA with multiple comparisons (ns—p R 0.05). (D) UMAP visualization of snRNA-seq of 12-week in vivo-grown tHIOs in 10ng/mL of EREG (n = 1 sequencing run of one tHIO). (E) Dot plot visualization for expression of canonical markers of neurons (S100B, PLP1, STMN2, and ELAVL4), endothelial cells (CDH5, KDR, ECSCR, and CLDN5), mesenchyme (VIM, COL1A1, COL1A2, and DCN), smooth muscle (ACTA2, TAGLN, ACTG2, and MYLK), epithelium (EPCAM, CDH1, CDX2, and CLDN4), immune cells (PTPRC, HLA-DRA, ARHGDIB, and CORO1A), and proliferative cells (MKI67 and TOP2A).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Epidermal Growth Factor (EGF) R&D Systems Cat#236-EG Epiregulin (EREG) R&D Systems Cat#1195-EP Recombinant Human Noggin-FC Purified from HEK293 cells expressing FC-tagged Noggin N/A Human R-Spondin1 Purified from Conditioned Medium from Cultrex HA-R-Spondin1-FC 293 T-Cells Cat# 3710-001-01 B27 Supplement Thermo Fisher Cat#17504044 HEPES Gibco Cat#15630080 GlutaMAX Gibco Cat#35050061 Penicillin-Streptomycin Thermo Fisher Cat#15140122 mTeSR 1 Stemcell Technologies Cat#85850 RPMI 1640 Thermo Fisher Cat#11875093 FBS Sigma Cat#12103C Dimethyl phenyl piperazinium Sigma Cat#D5891 NG-nitro-L-arginine methyl ester Sigma Cat#N5751 Atropine sulfate salt monohydrate Sigma Cat#A0132 Neurotoxin tetrodotoxin Tocris Cat#1078 M199 Gibco Cat#11150067 Heparin Sigma Cat#H3149-100KU FGF2 R&D Systems Cat#236-EG-01M N-acetylcysteine Sigma Cat#A9165-25G Accutase Corning Cat#MT2508CI Carbamyl-b-methylcholine chloride Sigma Cat#C5259 Scopolamine hydrobromide Tocris Cat#1414 Fibrinogen from bovine plasma Sigma Cat#F8630 Human Fibrinogen 1 Plasminogen Depleted Enzyme Research Lab Cat#FIB-1 X-Vivo 20 Lonza Cat#190995 Thrombin from bovine plasma Sigma Cat#T4648 StemSpan SFEM Stemcell Technologies Cat#9650 Knockout Serum Gibco Cat#10828010 Heparin Sigma Cat#H3149 FGF2 R&D Systems Cat#233-FB-MTO Aprotinin Sigma Cat#A6106 Critical commercial assays Neural Tissue Dissociation Kit Miltenyi Cat#130-092-628 103 Genomics Chromium Nuclei Isolation Kit 103 Genomics Cat#1000493 103 Chromium Controller v3 chemistry 103 Genomics Cat# 1000268 MagMax-96 Total RNA Isolation Kit/machine Thermo Fisher Cat#AM1830 SuperScript VILO cDNA Kit Thermo Fisher Cat#11754250 QuantiTect SYBR Green PCR Kit QIAGEN Cat#204145 MycoAlert Mycoplasma Detection Kit Lonza Cat#LT07-318 Proteome Profiler Human Phospho-Kinase Array Kit R&D Systems Cat#ARY003C Deposited data Raw scRNAseq data (human fetal intestine) Holloway et al.24 ArrayExpress: E-MTAB-9489 Raw scRNAseq data (HIO whole cell) This study ArrayExpress: E-MTAB-13463 Raw snRNAseq data (HIO and tHIO nuclei) This study ArrayExpress: E-MTAB-13469 Experimental models: Cell lines H9 ESC WiCell NIH registry#0062, RRID: CVCL_9773, female iPSC WTC11 Coriell Institute RRID: CVCL_Y803, male (Continued on next page) e2 Cell Stem Cell 32, 1–12.e1–e9, April 3, 2025

Techniques: Transplantation Assay, Staining, In Vivo, Sequencing, Expressing

Journal: Cell stem cell

Article Title: Coordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature.

doi: 10.1016/j.stem.2025.02.007

Figure Lengend Snippet: Figure 4. EREG-grown HIOs pattern blood vessels that are functional both in vitro and in vivo (A) Schematic of workflow for whole-mount imaging in vitro EREG-grown HIOs. (B) Representative IF staining of n = 4 different EREG-grown (10 ng/mL) HIOs for the presence of endothelial cells (PECAM; red) and DAPI (gray). All scale bars, 100 mm. (C) IF staining of EGF-grown tHIO (left) and EREG-grown tHIO (right), with stains for DAPI (gray), autofluorescent red blood cells in the 488-laser channel (red), and human-specific PECAM antibody (yellow). All scale bars, 50 mm. (D) Schematic of workflow for in vivo EREG-grown tHIO functionality test for connection with host vasculature. (E) Representative whole-mount IF staining of EGF-grown (10 ng/mL) tHIOs and EREG-grown (10 ng/mL) tHIOs for the presence of human endothelial cells (hsPECAM; red), HIO mCherry tag (white), lectin dye administered through tail vein injection (yellow), and DAPI (blue). All scale bars, 100 mm. (F) Quantification of flow cytometry analysis to quantify the percentage of hsPECAM+/lectin+ cells. Three 12-week-old tHIOs per condition were pooled per condition to ensure adequate material for the experiment. (G) Schematic of workflow for in vitro EREG-grown HIO functionality test using RVEC microfluidic device. (H) Representative IF staining of the RVECs (red) connecting to HIOs (gray) by visualizing VE-CAD-conjugated antibody (yellow) after flow through device. Any tube marked by yellow only is an endogenous HIO EC that has formed a connection with RVECs (white arrows). See also Videos S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Epidermal Growth Factor (EGF) R&D Systems Cat#236-EG Epiregulin (EREG) R&D Systems Cat#1195-EP Recombinant Human Noggin-FC Purified from HEK293 cells expressing FC-tagged Noggin N/A Human R-Spondin1 Purified from Conditioned Medium from Cultrex HA-R-Spondin1-FC 293 T-Cells Cat# 3710-001-01 B27 Supplement Thermo Fisher Cat#17504044 HEPES Gibco Cat#15630080 GlutaMAX Gibco Cat#35050061 Penicillin-Streptomycin Thermo Fisher Cat#15140122 mTeSR 1 Stemcell Technologies Cat#85850 RPMI 1640 Thermo Fisher Cat#11875093 FBS Sigma Cat#12103C Dimethyl phenyl piperazinium Sigma Cat#D5891 NG-nitro-L-arginine methyl ester Sigma Cat#N5751 Atropine sulfate salt monohydrate Sigma Cat#A0132 Neurotoxin tetrodotoxin Tocris Cat#1078 M199 Gibco Cat#11150067 Heparin Sigma Cat#H3149-100KU FGF2 R&D Systems Cat#236-EG-01M N-acetylcysteine Sigma Cat#A9165-25G Accutase Corning Cat#MT2508CI Carbamyl-b-methylcholine chloride Sigma Cat#C5259 Scopolamine hydrobromide Tocris Cat#1414 Fibrinogen from bovine plasma Sigma Cat#F8630 Human Fibrinogen 1 Plasminogen Depleted Enzyme Research Lab Cat#FIB-1 X-Vivo 20 Lonza Cat#190995 Thrombin from bovine plasma Sigma Cat#T4648 StemSpan SFEM Stemcell Technologies Cat#9650 Knockout Serum Gibco Cat#10828010 Heparin Sigma Cat#H3149 FGF2 R&D Systems Cat#233-FB-MTO Aprotinin Sigma Cat#A6106 Critical commercial assays Neural Tissue Dissociation Kit Miltenyi Cat#130-092-628 103 Genomics Chromium Nuclei Isolation Kit 103 Genomics Cat#1000493 103 Chromium Controller v3 chemistry 103 Genomics Cat# 1000268 MagMax-96 Total RNA Isolation Kit/machine Thermo Fisher Cat#AM1830 SuperScript VILO cDNA Kit Thermo Fisher Cat#11754250 QuantiTect SYBR Green PCR Kit QIAGEN Cat#204145 MycoAlert Mycoplasma Detection Kit Lonza Cat#LT07-318 Proteome Profiler Human Phospho-Kinase Array Kit R&D Systems Cat#ARY003C Deposited data Raw scRNAseq data (human fetal intestine) Holloway et al.24 ArrayExpress: E-MTAB-9489 Raw scRNAseq data (HIO whole cell) This study ArrayExpress: E-MTAB-13463 Raw snRNAseq data (HIO and tHIO nuclei) This study ArrayExpress: E-MTAB-13469 Experimental models: Cell lines H9 ESC WiCell NIH registry#0062, RRID: CVCL_9773, female iPSC WTC11 Coriell Institute RRID: CVCL_Y803, male (Continued on next page) e2 Cell Stem Cell 32, 1–12.e1–e9, April 3, 2025

Techniques: Functional Assay, In Vitro, In Vivo, Imaging, Staining, Injection, Cytometry

Journal: Neuro-Oncology Advances

Article Title: Transcriptomic analysis of EGFR co-expression and activation in glioblastoma reveals associations with its ligands

doi: 10.1093/noajnl/vdae229

Figure Lengend Snippet: (A) Ordered VST normalized expression of BTC (left) and EREG (right) on the TCGA-GBM (top) and Intellance-2 datasets (bottom). Mutation statuses are indicated underneath. (B) Boruta Z -scores of EGFR ligands ( TGFA , HBEGF , EREG , EGF , BTC , and AREG ) from 90 models built on the Intellance-2 and TCGA-GBM. For each model, Boruta’s decision to consider genes’ contribution significant is indicated. (C) EGFR ligand expression in neurons (NE), oligodendrocytes (OD), tumor cells (T), (tumor-associated) macrophages/microglia (TAM/MG), and astrocytes (AC) across multiple sc/sn-RNA-seq datasets. (D) Expression levels of the neuron marker RBFOX3 , inhibitory and excitatory neuron markers, and BTC in the Bolleboom-Gao snRNA-seq dataset. Abbreviations: EGFR, epidermal growth factor receptor; VST, variance-stabilizing transformation.

Article Snippet: Afterward, cells were stimulated (200 ng/mL, diluted in culture media) with EGF (GibcoTM Human EGF Recombinant Protein, PHG0311, Fisher Scientific), BTC (Betacellulin human, B3670, Sigma-Aldrich), EREG (Recombinant Human Epiregulin Protein, 1195-EP-025/CF, R&D Systems), or PBS as negative control.

Techniques: Expressing, Mutagenesis, RNA Sequencing, Marker, Transformation Assay

Journal: Neuro-Oncology Advances

Article Title: Transcriptomic analysis of EGFR co-expression and activation in glioblastoma reveals associations with its ligands

doi: 10.1093/noajnl/vdae229

Figure Lengend Snippet: (A) Representative confocal microscopic images of EGFR at multiple time points (0 minutes, 15 minutes, and 2 hours) after stimulation with EREG, BTC, EGF, and PBS as negative control. The right panel displays zoomed-in images showing intracellular accumulation of EGFR (spots) after 15 minutes and 2 hours of ligand stimulation. (B) quantitative analysis of microscopic images by a multistep algorithm showing the PBS-normalized number of spots per nucleus for each condition. Results are averaged across 2 cell lines, with each experiment conducted in replicate. Abbreviation: EGFR, epidermal growth factor receptor.

Article Snippet: Afterward, cells were stimulated (200 ng/mL, diluted in culture media) with EGF (GibcoTM Human EGF Recombinant Protein, PHG0311, Fisher Scientific), BTC (Betacellulin human, B3670, Sigma-Aldrich), EREG (Recombinant Human Epiregulin Protein, 1195-EP-025/CF, R&D Systems), or PBS as negative control.

Techniques: Negative Control

Journal: Cell reports

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters in collective cell migration of epithelial cells

doi: 10.1016/j.celrep.2024.114986

Figure Lengend Snippet: (A) Schematic of EGFRL-ScNeo expressed at the cell membrane. (B) Structure of EGFRL-ScNeos. SP, signal peptide; Pro, propeptide; Sc, mScarlet; EGF, EGF domain; TM, transmembrane domain; Neo, mNeonGreen; VV, two valine residues; Ig, immunoglobulin-like domain. (C) xy confocal images of EGFRL-ScNeos. Scale bar, 10 μm. (D) mScarlet/mNeonGreen fluorescence ratio of the cell membrane. The bar graphs show the mean values. Each dot represents the average value for one experiment ( n > 100 cells/experiment). (E) Western blot analysis of total cell lysates of EGFRL-ScNeo-expressing cells. *Full-length EGFRL-ScNeo; **cytoplasmic domain with mNeonGreen. (F) The proportion of cleaved EGFRL-ScNeo in (E). The bar graphs show the mean values. Each dot indicates an independent experiment. (G) Western blot analysis of supernatants of EGFRL-ScNeo-expressing cells. (H) The production rates of EGFRL from a single EGFRL-ScNeo-expressing cell. The bar graphs show the mean values. Each dot indicates an independent experiment. (I) mScarlet/mNeonGreen ratio images of EREG-ScNeo-expressing MDCK cells upon treatment with 10 nM TPA or 10 μM marimastat . Scale bar, 20 μm. (J) ERK activity of MDCK-4KO cells expressing EKARrEV-NLS stimulated with the supernatant of MDCK-4KO cells expressing HBEGF-ScNeo. Scale bar, 50 μm. (K) Time course of ERK activity in MDCK-4KO-EKARrEV-NLS cells stimulated with supernatant from MDCK-4KO cells expressing EGFRL-ScNeo. Solid lines represent the means from two independent experiments ( n > 1,000 cells/experiment). (L) Maximum ERK activity from the time course shown in (K). The bar graphs show the mean values from three independent experiments. Each dot represents the average value for one experiment ( n > 1,000 cells/experiment). See also and .

Article Snippet: Recombinant human EREG , PeproTech , Cat# 100-04.

Techniques: Membrane, Fluorescence, Western Blot, Expressing, Activity Assay

Journal: Cell reports

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters in collective cell migration of epithelial cells

doi: 10.1016/j.celrep.2024.114986

Figure Lengend Snippet: (A) Schematic of the SLIPT system. (B) Time course of normalized FRET/CFP ratio for TSen stimulated with various m D cTMP concentrations. Values were normalized to the average pre-stimulation baseline (20 min). Solid lines and shaded areas represent means and SDs from three independent experiments ( n > 100 cells/experiment). (C) miRFP703 and mScarlet/mNeonGreen ratio images of cells expressing AREG-ScNeo and eDHFR-cRaf. Images are snapshots of . Scale bar, 20 μm. (D) Schematic of SLIPT-induced EGFRL shedding and ERK activity observation. (E) Representative time-lapse ERK activity images. The white area at the center of the 0 min image indicates the EREG-producer cells. Scale bar, 100 μm. (F) Representative time-lapse ERK activity images. Each ligand producer is located at the center of the image. Images are snapshots of . Scale bar, 100 μm. (G) The time of maximum ERK activity in receiver cells in (F) after m D cTMP addition is plotted against the distance from the center. Each dot indicates a single cell. (H) Velocities of ERK waves propagated from each EGFRL producer. Each dot indicates a single producer-cell population. The red bars represent the means from three independent experiments, depicted by the three colors ( n = 28 [EREG], 36 [AREG], 50 [TGF-α], 30 [HBEGF], and 23 [NRG1] producer-cell populations). p values were calculated by a two-sample unpaired t test. (I) Maximum radius of ERK waves propagated from each EGFRL producer. Data in (H) were used for the analysis. The red bars represent the means. p values were calculated by a two-sample unpaired t test. (J) Western blot analysis of the supernatant of each producer cell incubated with or without 10 μM m D cTMP. (K) The production rates of EGFRL from each producer cell in (J). The mScarlet intensities of HBEGF supernatant with m D cTMP were set as 1. See also and and and .

Article Snippet: Recombinant human EREG , PeproTech , Cat# 100-04.

Techniques: Expressing, Activity Assay, Western Blot, Incubation

Journal: Cell reports

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters in collective cell migration of epithelial cells

doi: 10.1016/j.celrep.2024.114986

Figure Lengend Snippet: (A) (Top) Schematic of TGF-α-EREG chimera. (Bottom) mNeonGreen xz images of EREG, TGF-α, and a TGF-α-EREG chimera. Scale bars, 10 μm. (B) The velocity of the ERK wave propagated from each producer. Each dot indicates a single producer-cell population. The red bars represent the means from two independent experiments, depicted by the two colors ( n = 30 [EREG], 26 [TGF-α], and 24 [TGF-α-EREG chimera] producer-cell populations). p values were calculated by a two-sample unpaired t test. (C) The velocity of the ERK wave propagated from each producer cell to WT or EGFR-overexpressing (O/E) receiver cells. Each dot indicates a single producer-cell population. The red bars represent the means from two independent experiments ( n = 35 [EREG, WT], 41 [EREG, EGFR O/E], 32 [HBEGF, WT], and 12 [HBEGF, EGFR O/E] producer-cell populations). p values were calculated by a two-sample unpaired t test. (D) Representative ERK activity images in MDCK-α−1-catenin KO receiver cells. Each EGFRL producer cell is located at the center. Images were acquired 30 min after m D cTMP addition . Scale bar, 100 μm. (E) Maximum radius of the ERK wave propagated from each EREG-producer cell to each receiver cell. Each dot indicates a single producer-cell population. The red bars represent the means from three independent experiments, depicted by the three colors ( n = 38 [WT] and 42 [quinKO] producer-cell populations). p values were calculated by a two-sample unpaired t test. (F) The velocity of the ERK wave propagated from each producer cell to each receiver cell. Each dot indicates a single producer-cell population. The red bars represent the means from two independent experiments ( n = 21 [EREG, WT], 21 [EREG, E-cadherin KO], 24 [EREG, p120-catenin KO], 18 [HBEGF, WT], 11 [HBEGF, E-cadherin KO], and 21 [HBEGF, p120-catenin KO] producer-cell populations). p values were calculated by two-sample unpaired t test. (G) Representative ERK activity images in MDCK-4KO-EKARrEV-NLS receiver cells. Each producer cell expressing eDHFR-cRaf is located in the white area. Images were acquired 20 min after m D cTMP addition. Scale bar, 50 μm. (H) Maximum radius of the ERK wave propagation in (G). Each dot indicates a single producer-cell population. The red bars represent the means. n = 23 (WT) or 25 (TKO) producer-cell populations from three independent experiments. n = 6 (4KO) producer-cell populations from two independent experiments. n = 11 (dEREG) producer-cell populations from a single experiment. p values were calculated by a two-sample unpaired t test. See also and .

Article Snippet: Recombinant human EREG , PeproTech , Cat# 100-04.

Techniques: Activity Assay, Expressing

Journal: Cell reports

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters in collective cell migration of epithelial cells

doi: 10.1016/j.celrep.2024.114986

Figure Lengend Snippet: (A) Schematic of the boundary assay. (B) Representative ERK activity images in receiver cells adjacent to each producer cell . m D cTMP was added at 0 min. Scale bar, 100 μm. (C) Receiver-cell displacement adjacent to each producer cell. The red lines show the mean values. Each dot represents the average of a single field of view. n > 1,000 cells from three independent experiments, depicted by the three colors. p values were calculated by a two-sample unpaired t test. (D) Representative FRET/CFP images of receiver cells expressing ERK, tyrosine kinases, or ROCK biosensors. White arrowheads indicate the location of EGFRL-producer cells. Images were acquired 32 min after m D cTMP addition . Scale bar, 100 μm. (E) ERK activity in 10 representative cells around EREG or HBEGF producers was plotted over time after m D cTMP addition. (F) FWHM of ERK activation in receiver cells. Each dot indicates a single receiver cell. n = 50 cells from a single experiment. See also and .

Article Snippet: Recombinant human EREG , PeproTech , Cat# 100-04.

Techniques: Activity Assay, Expressing, Activation Assay

Journal: Cell reports

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters in collective cell migration of epithelial cells

doi: 10.1016/j.celrep.2024.114986

Figure Lengend Snippet: (A) Schematic of the experiment. (B) MDCK-4KO receiver cells surrounding EREG-ScNeo or HBEGF-ScNeo producer-cells. Cells were fixed and stained with anti-EEA1 and anti-Rab7 antibodies. White circles and arrowheads indicate mScarlet-positive vesicles co-localized with EEA1 and Rab7, respectively. The gray area indicates the producer cells. Scale bar, 5 μm. (C) Fraction of mScarlet-positive vesicles co-localized with EEA1 or Rab7 from images in (B). The bar graphs show the mean values. Each dot represents the average of a single field of view. n = 2 fields of view from a single experiment. (D) MDCK-Erbock-ErbB1 receiver cells surrounding EREG-ScNeo or HBEGF-ScNeo producer cells. Cells were fixed and stained with anti-EEA1, anti-Rab7, and anti-RFP antibody. Scale bar, 5 μm. (E) The proportion of mScarlet-positive vesicles co-localized with Rab7 or EEA1 from experiments in (D). The bar graphs show the mean values. Each dot represents the average of a single field of view. n = 11 (EREG) or 9 (HBEGF) fields of view from three independent experiments.

Article Snippet: Recombinant human EREG , PeproTech , Cat# 100-04.

Techniques: Staining

Journal: Cell reports

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters in collective cell migration of epithelial cells

doi: 10.1016/j.celrep.2024.114986

Figure Lengend Snippet: (A) ERK activity images in migrating MDCK WT, dEREG, or dHBEGF cells. Scale bar, 200 μm. (B) Kymographs of ERK activity generated from time-lapse FRET/CFP ratio images. White arrowheads indicate the first ERK wave propagating from the leader cells. White arrows indicate ERK waves propagating from the leader cells 10 h after removing the confinement. (C) Length of ERK waves propagating from the leader cells 10 h after removing the confinement. Each dot indicates a single ERK wave. Each color represents data from a single experiment. The red bars represent the means. p value was calculated by a two-sample unpaired t test. (D) Representative images of single-cell trajectories 10 to 22 h after removing the confinement. Scale bar, 200 μm. (E) Displacement of MDCK cells at 10 to 22 h after removing the confinement. Each dot represents a single cell. n > 1,000 cells for each experiment. (F) Schematic of an in vivo imaging of ERK activity during wound healing of mouse ear skin expressing hyBRET-ERK-NLS. (G) Representative ERK activity images in WT or Ereg −/− mouse ear skin . White arrows indicate ERK waves propagating from the wound edge (right black arrow). Scale bar, 100 μm. (H) Kymographs of ERK activity generated from time-lapse FRET/CFP ratio images. White and black arrows indicate ERK waves propagating from the wound edge (0 μm). (I) Displacement of mouse skin basal layer cells in 3 h toward the wound edge. Each dot represents a single cell. n > 1,000 cells for each mouse. See also and .

Article Snippet: Recombinant human EREG , PeproTech , Cat# 100-04.

Techniques: Activity Assay, Generated, In Vivo Imaging, Expressing

Journal: Cell reports

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters in collective cell migration of epithelial cells

doi: 10.1016/j.celrep.2024.114986

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant human EREG , PeproTech , Cat# 100-04.

Techniques: Virus, Recombinant, Microscopy, Sequencing, Knock-Out, Plasmid Preparation, Software