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(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 <t>and</t> <t>EREG</t> 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) <t>EGFR</t> 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).

Ligand For Egfr, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/recombinant+human+epiregulin/Recombinant+Human+Epiregulin+Protein/bio_rxiv__64898__2026__04__07__717082-73-23-26
Average 93 stars, based on 25 article reviews

ligand for egfr - by Bioz Stars, 2026-09

93/100 stars

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1) Product Images from "Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin"

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

Journal: bioRxiv

doi: 10.64898/2026.04.07.717082

Figure Legend 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).

Techniques Used: Staining, Immunohistochemical staining, Expressing, Immunostaining

(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).

Figure Legend 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).

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

Recombinant:

Article Title: Stabilization of EREG via STT3B-mediated N-glycosylation is critical for PDL1 upregulation and immune evasion in head and neck squamous cell carcinoma
Article Snippet: .. Recombinant human epiregulin, PNGase F, Endo H, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA). .. Tunicamycin (TM), carboxylic acid (JQ-1), Bix, AG490 were purchased from MCE (USA).

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy
Article Snippet: .. Recombinant human epiregulin, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA). .. An anti-GAPDH antibody was obtained from Santa Cruz Biotechnology (CA, USA).

Article Title: Overexpression of cyclooxygenase-2 (COX-2) in the mouse urinary bladder induces the expression of immune- and cell proliferation-related genes.
Article Snippet: .. Recombinant human epiregulin was purchased from R&D Systems (Minneapolis, MN). .. TRIzol1 reagent was purchased from Invitrogen (Carlsbad, CA).

Article Title: Humanized anti-epiregulin antibody, and cancer therapeutic agent comprising said antibody as active ingredient
Article Snippet: Fifteen micrograms of the linearized hEGFR expression vector (pCXZD1/EGFR#3) obtained by PvuI digestion was transfected into Ba/F3 cells by electroporation (Gene Pulser; BioRad) under conditions of 0.33 kV and 950 μFD. .. Transfected cells were selected in an RPMI1640 medium containing 10% FBS, 300 μg/mL Zeocin, and recombinant human Epiregulin (R&D Systems, Cat: 1195-EP/CF, 200 ng/mL); and the EGFR_BAF cell line was isolated. (2) Activity of Anti-Epiregulin Antibodies to Neutralize Human Epiregulin- or Monkey Epiregulin-Dependent Cell Proliferation of the EGFR_BAF Cell Line Experiments to measure the activity of anti-Epiregulin antibodies to neutralize human Epiregulin- or monkey Epiregulin-dependent cell proliferation were performed using the EGFR_BAF cell line isolated by the method described in (1). ..

Article Title: Autocrine epiregulin activates EGFR pathway for lung metastasis via EMT in salivary adenoid cystic carcinoma
Article Snippet: Cells were cultured in RPMI-1640 (Gibco BRL, Grand Island, NY) with 10% fetal bovine serum (Gibco) and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C. .. Recombinant Human epiregulin was obtained from R & D Systems (Minneapolis, MN, USA). .. Antibodies against GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA).

Transfection:

Isolation:

Activity Assay:

Image Search Results

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).

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

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.

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

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

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

Techniques: Generated

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.

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

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).

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).

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

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.

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.

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

(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.

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

(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.

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.

Techniques: Negative Control

$a - b , Gene-expression profiles of genes associated with adult and repair-induced stem cells in ileum organoids as in Fig. ( a ), or in primary intestinal epithelial cells as in Fig. ( b ). Dot color relates to mean expression values and dot size relates to fraction of expressing cells. a , right panel - dot color indicates log normalized expression. a , n = 953 cells; b , n = 15,184 single cells. c - d , Flow cytometric quantification of KIT + cell frequency ( c ) and qPCR quantification of tuft cell genes ( d ) in DOX-triggered POU2F3 overexpression organoids differentiated in tuft cell medium. Each dot is a well. n = 4 (c), 2 (d) wells per condition. One of 2 ( d ) or 3 ( c ) independent experiments on the same donor with similar results are shown (Supplementary Fig. ). e , Representative image (left) and quantification of organoid area (right) from POU2F3 overexpression clonal organoids, with or without DOX inducement. Each dot is an individual organoid, n = 46 (DOX - ) or 50 (DOX + ) organoids. Experiments were performed on 2 donors (Supplementary Fig. ). f - g , Representative flow cytometric analysis ( f ) and fluorescence image ( g ) of AVIL-lineage tracing organoids after irradiation. 3 independent experiments were performed on the same donor with similar results. h , qPCR quantification of EREG expression in sorted AVIL − and AVIL + cells from human ileum organoids. Each dot is a well, n = 3 (Diff) or 4 (Diff+IL-4/13) biologically replicates. Results are pooled from 2 independent experiments (Supplementary Fig. ), i , Genotype of human ileum EREG knock out organoids. j - k , Images ( j ) and quantification of organoid area ( k ) from WT and EREG −/− organoids exposed to IL-4/13 after irradiation (as in Fig. ). k , Results are pooled from 2 independent experiments, n = 900 (WT control), 700 ( EREG −/− control), 700 (WT irradiation) and 600 ( EREG −/− irradiation) individual organoids. l - n , WT and POU2F3 −/− organoids were differentiated for 7 days in tuft cell differentiation medium with IL-4/IL-13, passaged, then cultured for 7 days in human intestinal expansion medium by removal of EGF, with or without recombinant EREG (rEREG). Shown are representative images ( l ), quantification of organoid numbers ( m ), and organoid areas ( n ). Three independent experiments were performed on 2 donors (Supplementary Fig. ). m , Each dot is a well. n = 3 wells per condition. n , n = 40 (WT control), 20 ( POU2F3 −/− control), 40 (WT rEREG) and 40 ( POU2F3 −/− rEREG) individual organoids. e , g , j , l , Scale bar, 1 mm ( e , j , l ), 20 µm ( g ). c , d , h , m , Data are presented as mean values +/− standard error ( c , h , m ) or as mean values ( d ). e , k , n Boxplots show data from the 25th–75th percentile and whiskers extending to the minimum and maximum within 1.5 × inter-quartile range, with dots marking outliers. c , e , h , k , m , n , P values are derived from two-tailed Student’s t-test ( c , e , h , k , n ), or two-tailed Mann-Whitney test ( m ). Diff: human tuft cell differentiation medium; WT: wildtype; TA: Transit-Amplifying Cells; EEC: Enteroendocrine cell; DOX: doxycycline.$

Journal: Nature

Article Title: Tuft cells act as regenerative stem cells in the human intestine

doi: 10.1038/s41586-024-07952-6

Figure Lengend Snippet: a - b , Gene-expression profiles of genes associated with adult and repair-induced stem cells in ileum organoids as in Fig. ( a ), or in primary intestinal epithelial cells as in Fig. ( b ). Dot color relates to mean expression values and dot size relates to fraction of expressing cells. a , right panel - dot color indicates log normalized expression. a , n = 953 cells; b , n = 15,184 single cells. c - d , Flow cytometric quantification of KIT + cell frequency ( c ) and qPCR quantification of tuft cell genes ( d ) in DOX-triggered POU2F3 overexpression organoids differentiated in tuft cell medium. Each dot is a well. n = 4 (c), 2 (d) wells per condition. One of 2 ( d ) or 3 ( c ) independent experiments on the same donor with similar results are shown (Supplementary Fig. ). e , Representative image (left) and quantification of organoid area (right) from POU2F3 overexpression clonal organoids, with or without DOX inducement. Each dot is an individual organoid, n = 46 (DOX - ) or 50 (DOX + ) organoids. Experiments were performed on 2 donors (Supplementary Fig. ). f - g , Representative flow cytometric analysis ( f ) and fluorescence image ( g ) of AVIL-lineage tracing organoids after irradiation. 3 independent experiments were performed on the same donor with similar results. h , qPCR quantification of EREG expression in sorted AVIL − and AVIL + cells from human ileum organoids. Each dot is a well, n = 3 (Diff) or 4 (Diff+IL-4/13) biologically replicates. Results are pooled from 2 independent experiments (Supplementary Fig. ), i , Genotype of human ileum EREG knock out organoids. j - k , Images ( j ) and quantification of organoid area ( k ) from WT and EREG −/− organoids exposed to IL-4/13 after irradiation (as in Fig. ). k , Results are pooled from 2 independent experiments, n = 900 (WT control), 700 ( EREG −/− control), 700 (WT irradiation) and 600 ( EREG −/− irradiation) individual organoids. l - n , WT and POU2F3 −/− organoids were differentiated for 7 days in tuft cell differentiation medium with IL-4/IL-13, passaged, then cultured for 7 days in human intestinal expansion medium by removal of EGF, with or without recombinant EREG (rEREG). Shown are representative images ( l ), quantification of organoid numbers ( m ), and organoid areas ( n ). Three independent experiments were performed on 2 donors (Supplementary Fig. ). m , Each dot is a well. n = 3 wells per condition. n , n = 40 (WT control), 20 ( POU2F3 −/− control), 40 (WT rEREG) and 40 ( POU2F3 −/− rEREG) individual organoids. e , g , j , l , Scale bar, 1 mm ( e , j , l ), 20 µm ( g ). c , d , h , m , Data are presented as mean values +/− standard error ( c , h , m ) or as mean values ( d ). e , k , n Boxplots show data from the 25th–75th percentile and whiskers extending to the minimum and maximum within 1.5 × inter-quartile range, with dots marking outliers. c , e , h , k , m , n , P values are derived from two-tailed Student’s t-test ( c , e , h , k , n ), or two-tailed Mann-Whitney test ( m ). Diff: human tuft cell differentiation medium; WT: wildtype; TA: Transit-Amplifying Cells; EEC: Enteroendocrine cell; DOX: doxycycline.

Article Snippet: For specific experiments, 10 ng ml −1 human IL-27 (Peprotech), 10 ng ml −1 human IL-25 (Peprotech), 10 ng ml −1 human SCF (Peprotech), 50 ng ml −1 recombinant human Epiregulin (Peprotech), were used.

Techniques: Expressing, Over Expression, Fluorescence, Irradiation, Knock-Out, Control, Cell Differentiation, Cell Culture, Recombinant, Derivative Assay, Two Tailed Test, MANN-WHITNEY

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