Journal: Bone

Article Title: The reduced osteogenic potential of Nf1 -deficient osteoprogenitors is EGFR-independent

doi: 10.1016/j.bone.2017.10.012

Figure Lengend Snippet: A: Ereg expression in WT and Nf1-deficient mBMSCs (qPCR, n=3). B: Epiregulin protein expression in WT and Nf1-deficient mBMSCs (Western blot, n=3, Right graph: densitometric analysis). C: Egfr expression in WT and Nf1-deficient mBMSCs (qPCR, n=3). D: EGFR protein expression in WT and Nf1 deficient mBMSCs (Western blot, n=3, Right graph: densitometric analysis). E: Level of phosphorylated EGFR (p-EGFR), EGFR and β-actin in A431 cells treated with the conditioned medium (CM) from WT (grey bar) and Nf1-deficient (KO, black bar) mBMSCs in the presence of IgG control or an epiregulin neutralizing antibody (Western blot, n=3, Right graph: densitometric analysis). * and #: p<0.05 between genotypes and treatments, respectively. qPCR gene expression is normalized by Hprt expression.

Article Snippet: Cells were then treated with the conditioned media plus normal goat IgG control (AB-108-C, R&D Systems) or Epiregulin neutralizing antibody (AF1068-SP, R&D Systems) at the final concentration of 0.4 μg/ml.

Techniques: Expressing, Western Blot, Control, Gene Expression

Journal: Bone

Article Title: The reduced osteogenic potential of Nf1 -deficient osteoprogenitors is EGFR-independent

doi: 10.1016/j.bone.2017.10.012

Figure Lengend Snippet: A–B, D–E and G, H: Expression of early osteoblast marker genes (Alpl, Ibsp) in response to EGFR or Epiregulin inhibition during osteogenic differentiation (A–B: AG-1478, D–E: Poziotinib and G, H: epiregulin-neutralizing antibody) in WT and Nf1-deficient (KO) mBMSCs (qPCR, n=3, * and #: p<0.05 between genotypes and treatments, respectively). C, F and I: ALP activity in response to AG-1478, Poziotinib and Anti-Ereg neutralizing antibodies, respectively (n=3, * and #: p<0.05 between genotypes and treatments, respectively). qPCR gene expression is normalized by Hprt expression.

Article Snippet: Cells were then treated with the conditioned media plus normal goat IgG control (AB-108-C, R&D Systems) or Epiregulin neutralizing antibody (AF1068-SP, R&D Systems) at the final concentration of 0.4 μg/ml.

Techniques: Expressing, Marker, Inhibition, Activity Assay, Gene Expression

Journal: Middle East Fertility Society Journal

Article Title: Epiregulin dysregulation in polycystic ovary syndrome: metabolic and ovarian implications

doi: 10.1186/s43043-025-00226-9

Figure Lengend Snippet: Fig. 2 Correlation between estradiol (pg/mL) and epiregulin (pg/mL) in women with PCOS (n = 60)

Article Snippet: Serum epiregulin levels were quantified using a Human Epiregulin ELISA Kit (R&D Systems, USA), following standard ELISA procedures, with optical density measured at 450 nm using a microplate reader (BioTek ELx800, Agilent, USA).

Techniques:

Journal: Middle East Fertility Society Journal

Article Title: Epiregulin dysregulation in polycystic ovary syndrome: metabolic and ovarian implications

doi: 10.1186/s43043-025-00226-9

Figure Lengend Snippet: Fig. 1 Correlation between testosterone (ng/mL) and epiregulin (pg/mL) in women with PCOS (n = 60)

Article Snippet: Serum epiregulin levels were quantified using a Human Epiregulin ELISA Kit (R&D Systems, USA), following standard ELISA procedures, with optical density measured at 450 nm using a microplate reader (BioTek ELx800, Agilent, USA).

Techniques:

Journal: Biology of reproduction

Article Title: Effect of epidermal growth factor-like peptides on the metabolism of in vitro- matured mouse oocytes and cumulus cells.

doi: 10.1095/biolreprod.113.115311

Figure Lengend Snippet: FIG. 3. Effect of FSH, EGF, and EGF-like peptides on COC b-O-linked glycosylation. A1) Protein b-O-linked glycosylation was examined at 12 h IVM in the presence of control (no treatment), FSH, AREG, EREG, BTC, or EGF and b-O-linked glycosylation (CTD110.6) and nuclear staining (PI) fluorescence were imaged. Images shown are representative of 30 COCs per treatment group over three replicate experiments. Original magnification 360. A2) Quantification of relative CTD110.6 fluorescence in cumulus cells and oocytes. B) COC mRNA expression of Ogt was measured at 6 h IVM (n ¼ 6). Bars not sharing a common letter are significantly different (P , 0.02). The data represent means 6 SEM.

Article Snippet: COC IVM IVM COCs were cultured in bicarbonate buffered aMEM (Gibco) supplemented with 3 mg/ml BSA and either recombinant human FSH (50 mIU/ml; Puregon; Organon, Oss, The Netherlands), recombinant human EGF (10 ng/ml; R&D Systems, Minneapolis, MN), recombinant mouse AREG (50 ng/ml; R&D Systems), recombinant mouse EREG (50 ng/ml; R&D Systems), or recombinant mouse BTC (50 ng/ml; R&D Systems), at 378C with 5% CO 2 in air.

Techniques: Glycoproteomics, Control, Staining, Fluorescence, Expressing

Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Graphical representations of serum concentrations of ligands measured by ELISA . Individual data of serum EREG, HGF, EGF, AREG, NRG, IGF-1 and TGF- α are summarized by graphs. Blue bars show serum levels at pre-treatment and red bars show those at progression disease.

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Survival curves of pretreatment hepatocyte growth factor (HGF) and epiregulin (EREG) levels among KRAS wild-type patients and all wild-type of KRAS , BRAF , PIK3CA and NRAS. Survival curves of PFS in terms of HGF levels are shown among ( A ) KRAS wild-type patients and ( B ) among all wild-type patients. Survival curves of PFS in terms of EREG levels are shown among ( C ) KRAS wild-type patients and ( D ) among all wild-type patients. Among KRAS wild-type patients, survival curves of OS in ( E ) HGF levels and ( F ) EREG levels are shown. Patients with low levels of ligands had longer OS compared with patients with high levels.

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques:

Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Results of serum concentration of hepatocyte growth factor (HGF) and epiregulin (EREG) and genomic mutations of KRAS , BRAF , PIK3CA and NRAS

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques: Concentration Assay

Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Objective response rate (ORR) and disease control rate (DCR) by pretreatment serum levels of hepatocyte growth factor (HGF) and epiregulin (EREG) when the cutoff values are median of serum concentration

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques: Control, Concentration Assay

Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Survival curves by the change of serum levels of ligands after treatment. Survival curves of PFS by pretreatment levels of HGF and EREG (high/low) and change of serum levels at PD compared with pretreatment (elevation/no elevation) levels are shown. ( A ) PFS curves divided by change of serum HGF levels. ( B ) PFS curves divided by change of serum EREG levels. ( C ) OS curves divided by change of serum HGF levels. ( D ) OS curves divided by change of serum EREG levels.

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques:

Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Objective response rate (ORR) and disease control rate (DCR) by pretreatment serum levels of hepatocyte growth factor (HGF) and epiregulin (EREG) when the appropriate cutoff values were evaluated by ROC curve analysis

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques: Control

Journal: International Journal of Oral Science

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

doi: 10.1038/s41368-024-00311-1

Figure Lengend Snippet: Dysregulated EREG up-regulates PDL1 expression in human HNSCC cells. a Representative images of EREG staining in two patients with PDL1 and CD8 expression. Case 1 showed high expression of EREG with high expression of PDL1 and low expression of CD8. Case 2 showed low expression of EREG with low expression of PDL1 and high expression of CD8. b Hotmap of H-score of EREG and other immune-related markers in a cohort of 124 HNSCC tissues. Statistical analysis of IHC staining indicated that EREG expression is positively correlated with PDL1 ( R = 0.589 0, P < 0.001) and negatively correlated with EREG ( R = −0.186 5, P < 0.001) expression in HNSCC tissues. c Western blot analysis of PDL1 from four different HNSCC cell lines treated with 50 ng/mL epiregulin for 24 has indicated and HA-PDL1 from HEK293 cells treated with 50 ng/mL epiregulin for 24 h. d Western blot analysis of PDL1 expression in shControl and two independent shEREG stable clones of HN4 cells. e Cell surface analysis of PDL1 protein in three different HNSCC cell lines using flow cytometry

Article Snippet: Recombinant human epiregulin, PNGase F, Endo H, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Expressing, Staining, Immunohistochemistry, Western Blot, Clone Assay, Flow Cytometry

Journal: International Journal of Oral Science

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

doi: 10.1038/s41368-024-00311-1

Figure Lengend Snippet: EREG upregulates PDL1 through c-Myc. a Western blot analysis of PDL1 and c-Myc expression in HN6 and HN13 cells after treatment with EREG for different time intervals. b Plot of densitometry results for the levels of PDL1 and c-Myc. c CAL27 and HN13 cells were pretreated with various inhibitors for 1 h followed by stimulation with epiregulin for 24 h. The levels of PDL1 and c-Myc were examined by Western blot analysis. d Western blot analysis of PDL1, p-EGFR, EGFR, and GAPDH expression in CAL27 and HN13 cells pretreated with various EGFR inhibitors for 1 h followed by stimulation with epiregulin for 24 h. e Twenty-four hours after transfection with si-EGFR or si-NC short interfering RNAs (siRNAs), cells were further treated with EREG for 24 h, and the expression of PDL1, p-EGFR, EGFR, and GAPDH was detected by Western blot. f CAL27 and HN4 cells were pretreated with various BET BD inhibitors for 1 h followed by stimulation with epiregulin for 24 h. The levels of PDL1 and c-Myc were examined by Western blot analysis. g HN30 cells were treated with various BET BD inhibitors for 24 h, and the levels of PDL1 and c-Myc were examined by Western blot analysis

Article Snippet: Recombinant human epiregulin, PNGase F, Endo H, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Western Blot, Expressing, Transfection

Journal: International Journal of Oral Science

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

doi: 10.1038/s41368-024-00311-1

Figure Lengend Snippet: EREG is glycosylated in HNSCC cancer cells. a Expression of EREG protein in primary HNSCC patient samples and cell lines. Western blot analysis of EREG in 14 representative HNSCC patient samples and 9 HNSCC cell lines. b Western blot analysis of EREG expression in shControl and shEREG stable clones of HN4 and HN30 cells. c Western blot analysis of EREG expression in siNC and three individual small interfering RNAs (siRNAs) targeting EREG in both HN4 and HN30 cells. d Western blot analysis of EREG expression in HN4 cells with glucose supplementation (5.55 mmol/L) at the indicated times. HN4 cells were cultured in serum-free medium with a low concentration of glucose (5.55 mmol/L) for 24 h before glucose supplemention. e Glycosylation pattern of EREG protein in HN4, SCC9 and FaDu cells. Cell lysates were treated with PNGase F and analyzed by Western blot analysis. Black circles indicate glycosylated EREG, and arrowheads indicate non-glycosylated EREG. f Glycoprotein staining and Coomassie blue staining of PNGase F-treated purified EREG. Horseradish peroxidase (HRP) and soybean trypsin inhibitor (STI) served as positive and negative controls, respectively. g Cell lysates from the indicated cell lines were treated with PNGase F, Endo H, and O-glycanase for 1 h at 37 °C in vitro. h Immunoblot of EREG in HN4 cells treated with inhibitors blocking N-linked or O linked glycosylation as indicated. i Immunoblot of EREG in HN4 and HN30 cells treated with the N-linked glycosylation inhibitors TM as indicated. j Immunoblot of EREG in HEK293-EREG cells treated with the N-linked glycosylation inhibitor TM or the O-linked glycosylation inhibitor benzyl as indicated

Article Snippet: Recombinant human epiregulin, PNGase F, Endo H, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Expressing, Western Blot, Clone Assay, Cell Culture, Concentration Assay, Glycoproteomics, Staining, Purification, In Vitro, Blocking Assay

Journal: International Journal of Oral Science

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

doi: 10.1038/s41368-024-00311-1

Figure Lengend Snippet: N-glycosylation of EREG is critical for stabilization of EREG in HNSCC cells. Deglycosylation of EREG enhanced the turnover of EREG. HN4 ( a ) and 293-EREG ( b ) cells were treated with 10 μg/mL tunicamycin (N-glycosylation inhibitor) for 24 h followed by pulse-chase with 100 μg/mL cycloheximide. Protein levels at the indicated time points were evaluated by immunoblot analysis. The intensity of the glycosylated form versus the nonglycosylated form of EREG was quantified using ImageJ software. c Immunoblot of EREG in HN4 cells treated with CHX for the indicated time in the presence or absence of TM and MG132. d HEK293 cells were transfected with Flag-EREG in the presence or absence of MG132 and/or TM. Flag-EREG was then immunoprecipitated followed by immunoblotting using anti-ubiquitin antibody. e Schematic diagram of EREG amino acid sequence alignment among different species. The NXT motif is shown in blue. f Predicted N-glycosylation sites of human EREG by NetNGlyc1.0 Server. g Western blot analysis of the protein expression pattern of EREG WT and its NQ mutants. The nonglycosylated form in Lane 13 indicates EREG-WT with overnight treatment with TM. h Western blot analysis of the protein expression pattern of EREG WT and its NQ mutants. Cell lysates were treated with PNGase F and analyzed by Western blot. i The indicated cell lines were treated with CHX at the indicated intervals. The intensity of EREG protein was quantified using ImageJ software. j Ubiquitination of EREG proteins in EREG-WT- or EREG-5NQ mutant-expressing HEK293 cells. EREG proteins were immunoprecipitated with HA antibody and then immunoblotted with ubiquitin antibody

Article Snippet: Recombinant human epiregulin, PNGase F, Endo H, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Glycoproteomics, Pulse Chase, Western Blot, Software, Transfection, Immunoprecipitation, Ubiquitin Proteomics, Sequencing, Expressing, Mutagenesis

Journal: International Journal of Oral Science

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

doi: 10.1038/s41368-024-00311-1

Figure Lengend Snippet: Glycosylation of EREG is crucial for maintaining its membrane subcellular location and autocrine activity. a GFP-EREG localization in HEK293 cells expressing WT, WT + TM, N47Q, or 5NQ mutant EREG by IF staining. b Colocalization of EREG and F-actin in HEK293 cells expressing WT, 5NQ, or N90Q mutant EREG by IF staining. c ELISA of EREG levels in conditioned medium from HEK293 cells expressing WT, N47Q, N90Q, or 2NQ mutant EREG. d HN4 cells were treated for 5 min with CM from HEK293 cells expressing vector, WT, or 5NQ mutant and analyzed by WB with the indicated antibodies. e Protein structure of mutant N47Q. The N and O atoms of the polar uncharged asparagine at position 47 of the wild-type protein form hydrogen bonds with the polar uncharged serine O atom at position 44 and the polar uncharged asparagine O atom at position 90. The hydrogen bond distances are 2.0 and 1.9 Å. The N47Q mutation results in the substitution of the polar uncharged asparagine amino acid at position 47 by the polar uncharged glutamine. After the mutation to glutamine, it forms a hydrogen bond with the non-polar serine O atom at position 44. The hydrogen bond distance is 3.1 Å, and it cannot interact with the N90 amino acid in polarity. The changes in the interaction of these amino acids may lead to changes in the protein structure and functional components after the mutation

Article Snippet: Recombinant human epiregulin, PNGase F, Endo H, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Glycoproteomics, Membrane, Activity Assay, Expressing, Mutagenesis, Staining, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Functional Assay

Journal: International Journal of Oral Science

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

doi: 10.1038/s41368-024-00311-1

Figure Lengend Snippet: The glycosylation of EREG is induced by STT3B. a HA-EREG was co-expressed with increasing amounts of Flag-STT3B in HEK293 cells. Lysates were subjected to Western blot analysis. b Western blot analysis of STT3B, EREG and PDL1 expression in HN4 cells after transfection with siSTT3B or siNC siRNAs. c HN4 cells were transfected with control or STT3B siRNA. After cells being treated with CHX, the expression of endogenous EREG was analyzed by Western blotting. d HEK293 cells were transiently co-transfected with Flag-STT3B and HA-EREG. Cell extracts were immunoprecipitated separately with anti-Flag or anti-HA antibodies, and the associated EREG and STT3B proteins were examined by Western blotting. Red pentagram symbol indicates glycosylated EREG, blue pentagram symbol indicates un-glycosylated EREG. e Endogenous EREG and STT3B were immunoprecipitated from HN4 cells, and bound endogenous STT3B and EREG were examined by Western blotting. Red pentagram symbol indicates glycosylated EREG, blue pentagram symbol indicates un-glycosylated EREG. f HEK293 cells were transiently cotransfected with Flag-STT3B and different HA-EREG mutants (WT, N47Q, N90Q, N146Q, 2NQ, and 5NQ). Cell extracts were immunoprecipitated separately with anti-Flag antibodies, and the associated EREG proteins were examined by Western blotting. Red pentagram symbol indicates glycosylated EREG, blue pentagram symbol indicates un-glycosylated EREG. g Representative paired immunohistochemistry staining of EREG, STT3B and PDL1. h Statistical analysis of immunohistochemistry staining of the tissue array showed that both EREG and PDL1 expression are positively correlated with STT3B expression in HNSCC ( P < 0.001)

Article Snippet: Recombinant human epiregulin, PNGase F, Endo H, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Glycoproteomics, Western Blot, Expressing, Transfection, Control, Immunoprecipitation, Immunohistochemistry, Staining

Journal: International Journal of Oral Science

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

doi: 10.1038/s41368-024-00311-1

Figure Lengend Snippet: STT3B inhibitor suppresses EREG glycosylation and enhances the efficacy of PD-1 blockade in vivo. a HN4 cells were treated with 0-20 μmol/L NGI-1 (OST inhibitor) for 24 h. The expression of EREG was examined by immunoblotting. b HN4 cells were treated with or without NGI-1 followed by treatment with CHX, and the expression of endogenous EREG was analyzed by Western blotting. c GFP-EREG localization in HEK293 cells treated with NGI-1 or TM by IF staining. d , e T cell-mediated cytotoxicity was assessed in MTCQ1 cells with or without the NGI-1 treatment (n = 5). ns, not significant; * P < 0.05; ** P < 0.01. f Mouse MTCQ1 cells were injected into C57 mice. When established tumors were palpable, the mice were treated with vehicle, NGI-1, anti-PDL1 mAb, or NGI-1 + anti-PDL1 mAb (n = 5) via i.p. injection. MTCQ1 tumors in each group were harvested and photographed at the end of the experiment. Photographs of the xenograft tumors are shown. g Tumor weights were measured for each treatment group at autopsy. h Tumors were measured with calipers, and values were plotted. The vertical bars indicate the mean tumor size (mm 3 ) ± SE. i IHC scores for Ki-67 expression in tumor sections from each treatment group. (* P < 0.05, ** P < 0.01, and *** P < 0.001). j IHC scores for CD8 expression in tumor sections from each treatment group. (* P < 0.05, ** P < 0.01). k Quantitative analysis of TUNEL-positive cells among groups. (n = 5, * P < 0.05)

Article Snippet: Recombinant human epiregulin, PNGase F, Endo H, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Glycoproteomics, In Vivo, Expressing, Western Blot, Staining, Injection, TUNEL Assay

Journal: International Journal of Oral Science

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

doi: 10.1038/s41368-024-00311-1

Figure Lengend Snippet: The proposed model of STT3B-mediated EREG glycosylation in promotion of immunoevasion via PDL1 upregulation in HNSCC

Article Snippet: Recombinant human epiregulin, PNGase F, Endo H, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Glycoproteomics

Journal: Cell reports

Article Title: Disease-association analysis of an inflammation-related feedback loop.

doi: 10.1016/j.celrep.2013.01.028

Figure Lengend Snippet: Figure 1. Scheme for Screening Positive Regulators of IL-6-Mediated Amplification and Inflammation (A) Schematic of the primary screen. A BC1 mouse type 1 collagen+ cell line was cultured in 96-well plates and treated with a lentivirus that encoded shRNA specific for candidate genes. The resulting BC1 cells were stimulated with human IL-6, soluble IL-6 receptor, and mouse IL-17. Mouse IL-6 concentrations in the supernatant and cell survival were measured by ELISA and mitochondrial activity, respectively. (B) Top: Mouse IL-6 expression in BC1 cells stimulated with various concentrations of human IL-6, human soluble IL-6 receptor, and mouse IL-17. Bottom: Survival of BC1 cells transduced with control shRNA after stimulation with various concentrations of human IL-6, human soluble IL-6 receptor, and mouse IL-17. (C and D) Candidate genes were selected based on two criteria: (i) expression of mouse IL-6, and (ii) cell survival. We selected shRNA that resulted in mouse IL-6 expression levels that were less than 35% the average IL-6 expression level in the 96-well plate. Cell survival was also evaluated based on mitochondrial activity using TCO reagent (gray squares). The mean and SD for all BC1 cells were 1.88 and 0.08, respectively. The threshold value was therefore set at 1.88 0.08 = 1.8. shRNA-screening results specific for 11 known genes in the IL-6 (C) and IL-17 (D) signaling pathways are shown. Mouse IL-6 expression levels are represented by the black bars; relative cell survival by the gray squares. Black and white diamonds on the bottom denote shRNA that fulfilled the primary screening criteria by more than 65% and inhibited IL-6 production by more than 50%, respectively. Dashed lines indicate thresholds (65%, 50%, and 1.8). See also Figures S1, S2, S3, S4, S5, S6, and Table S10.

Article Snippet: Mouse Epiregulin ELISA Kit, Human soluble IL-6 receptor a, mouse IL-23, human epiregulin, mouse epiregulin, and anti-mouse epiregulin antibody were obtained from (R&D Systems).

Techniques: Cell Culture, shRNA, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Transduction, Control, Protein-Protein interactions

Journal: Cell reports

Article Title: Disease-association analysis of an inflammation-related feedback loop.

doi: 10.1016/j.celrep.2013.01.028

Figure Lengend Snippet: Figure 2. The Epiregulin-ErbB1 Pathway Activates the IL-6 Amplifier In Vitro (A) IL-6 amplifier activation was suppressed in the absence of FBS. BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 for 24 hr in the absence or presence of 10% FBS. Gray dots indicate cell survival values monitored by TCO reagent. Left: Culture supernatants were collected and assessed using an ELISA specific for mouse IL-6. Cell survival was also evaluated based on mitochondrial activity (gray squares; all points were above the threshold value). Right: Il-6 mRNA expression 3 hr after stimulation was evaluated using real-time PCR. (B) BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 3 hr. Epiregulin (Ereg) expression was then evaluated using real-time PCR. (C) BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 48 hr with or without EGF, another ErbB1 ligand. Culture supernatants were collected and assessed using an ELISA specific for epiregulin. (D) BC1 cells were stimulated with epiregulin in the absence of FBS for 3 hr. Epiregulin (Ereg) expression was evaluated using real-time PCR. (E) BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 3 hr. ErbB1 (Egfr) expression was then evaluated using real-time PCR. (F) BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 24 hr with or without various concentrations of epiregulin. Culture supernatants were collected and assessed using ELISA specific for IL-6. (G–I) Il-6 (G), Ccl20 (H), and IkBz (I) mRNA expressions in BC1 cells 3 hr (G and H) or 1 hr (I) after stimulation with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS with or without epiregulin were evaluated using real-time PCR. (J) BC1 cells were treated with a lentivirus encoding shRNA specific for epiregulin (Ereg) and cultured in the presence of puromycin. The resulting cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 48 hr. Culture supernatants were collected and assessed using ELISA specific for mouse IL-6. Mean scores ± SD are shown. p values were calculated using Student’s t tests. * and #, p < 0.05; ** and ##, p < 0.01; ***p < 0.001. See also Figure S7 and Table S10.

Article Snippet: Mouse Epiregulin ELISA Kit, Human soluble IL-6 receptor a, mouse IL-23, human epiregulin, mouse epiregulin, and anti-mouse epiregulin antibody were obtained from (R&D Systems).

Techniques: In Vitro, Activation Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, shRNA, Cell Culture

Journal: Cell reports

Article Title: Disease-association analysis of an inflammation-related feedback loop.

doi: 10.1016/j.celrep.2013.01.028

Figure Lengend Snippet: Figure 3. The Epiregulin Signal Enhances NFkB Activation via the PI3K a Pathway (A) MEF cells were stimulated with epiregulin in the presence or absence of IL-6 and IL-17 and then investigated for the phosphorylation of Akt, p65, STAT3, and ERK1/ERK2. (B) HeLa cells were stimulated with epiregulin in the presence or absence of IL-17 and then investigated for NFkB reporter activity using a 53 NFkB-luc construct. (C) BC1 cells were stimulated with epiregulin in the presence or absence of LY294002 and then investigated for NFkB reporter activity using a 53 NFkB-luc construct. (D) IKKa- and IKKg-deficient MEF cells as well as wild-type MEF cells (WT) were stimulated with epiregulin (100 ng/ml) in the presence or absence of human IL-6 plus soluble IL-6 receptor and/or IL-17 for 12 hr. Culture supernatants were collected and assessed using an ELISA specific for mouse IL-6. (E and F) BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS with or without epiregulin and various concentrations of PIK75 (E) or TGX221 (F) for 24 hr. Cell survival was also evaluated based on mitochondrial activity. Culture supernatants were collected and assessed using an ELISA specific for mouse IL-6. (G and H) (G) BC1 cells were treated with a lentivirus encoding shRNA specific for Pik3ca and stimulated with epiregulin (100 ng/ml) in the presence or absence of human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 24 hr. Culture supernatants were collected and assessed using an ELISA specific for mouse IL-6. (H) Pik3ca expression was also evaluated using real-time PCR. Mean scores ± SD are shown. p Values were calculated using Student’s t tests. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant. See also Figure S9 and Table S10.

Article Snippet: Mouse Epiregulin ELISA Kit, Human soluble IL-6 receptor a, mouse IL-23, human epiregulin, mouse epiregulin, and anti-mouse epiregulin antibody were obtained from (R&D Systems).

Techniques: Activation Assay, Phospho-proteomics, Activity Assay, Construct, Enzyme-linked Immunosorbent Assay, shRNA, Expressing, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: Disease-association analysis of an inflammation-related feedback loop.

doi: 10.1016/j.celrep.2013.01.028

Figure Lengend Snippet: Figure 4. Epiregulin-ErbB1 Signaling Triggers Autoimmune Diseases in Mouse Models (A) IL-6 and IL-17 on days 6, 7, and 8 were injected into the joints of F759 mice in the presence or absence of joint injections of lentivirus encoding shRNA specific for ErbB1 (Egfr) (n = 6), Ereg (n = 6), or a nontarget sequence (n = 6) on days 0, 2, and 4 followed by analysis of epiregulin and IL-6 expression in joint synovial tissues on day 15. (B) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 6, 7, and 8 and joint injections of lentivirus encoding shRNA specific for ErbB1 (Egfr) (open squares, open triangles, and crosses; n = 3), NFkB p65 (Rela) (asterisks; n = 3), or a nontarget sequence (diamonds; n = 3) on days 0, 2, and 4. (C) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 6, 7, and 8 and joint injections of a lentivirus encoding shRNA specific for epiregulin (Ereg) (crosses, asterisks, and open circles; n = 3), NFkB p65 (Rela) (open triangles; n = 3), or a nontarget sequence (squares; n = 3) on days 0, 2, and 4 and those from the left legs of F759 mice after left leg joint injections of saline alone (filled diamonds, n = 3). (D) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 0, 1, and 2 and joint injections of anti-epiregulin antibodies (1 mg) on days 0–23 (open squares, n = 3) and from the left legs of F759 mice after left leg joint injections of IL-17, IL-6, and control IgG (open circles, n = 3), or saline (open triangles, n = 3). (E) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 0, 1, and 2 and PD153035 (10 mg) on days 0–23 (triangles, n = 5) and from the left legs of F759 mice after left leg joint injections of IL-17, IL-6, and DMSO (squares, n = 5), or injections of DMSO only (diamonds, n = 5). (F) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 0, 1, and 2 and PD168393 (10 mg) on days 0–24 (triangles, n = 5) and from the left legs of F759 mice after left leg joint injections of IL-17, IL-6, and DMSO (squares, n = 5), or injections of DMSO only (diamonds, n = 5). (G) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 0, 1, and 2 and gefitinib (10 mg) on days 0–22 (triangles, n = 4) and from the left legs of F759 mice after left leg joint injections of IL-17, IL-6, and DMSO (squares, n = 4), or DMSO only (diamonds, n = 4). (H) Ankle joints from each mouse in (D) were fixed and embedded in paraffin. Histological analysis was performed using sections stained with hematoxylin-eosin. These experiments were performed at least three times independently; representative data are shown. (I) WT mice (2 months old) were intravenously injected with Th17 cells from WT mice with EAE and intraperitoneally injected with gefitinib (open squares, n = 5) or DMSO (open diamonds, n = 5) on days 0–6. (J) WT mice (2 months old) were intravenously injected with Th17 cells from WT mice with EAE and intraperitoneally injected with gefitinib (closed bars, n = 5) or DMSO (open bars, n = 5) on days 0–6. Mononuclear cells from the spinal cords of Th17-transferred C57BL/6 mice were isolated on day 13. The resulting cell populations

Article Snippet: Mouse Epiregulin ELISA Kit, Human soluble IL-6 receptor a, mouse IL-23, human epiregulin, mouse epiregulin, and anti-mouse epiregulin antibody were obtained from (R&D Systems).

Techniques: Injection, shRNA, Sequencing, Expressing, Saline, Control, Staining, Isolation

Journal: Cell reports

Article Title: Disease-association analysis of an inflammation-related feedback loop.

doi: 10.1016/j.celrep.2013.01.028

Figure Lengend Snippet: Figure 5. Epiregulin-ErbB1 Signaling Is Involved in Activation of the Human IL-6 Amplifier (A) Human synovial cell lines were stimulated for 3 hr with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS with or without epiregulin (100 ng/ml). Il-6 expression in the resulting cells was evaluated using real-time PCR. (B) Human synovial cells were stimulated for 3 hr with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the presence of FBS with or without PD153035 (10 mg/ml). Il-6 expression in the resulting cells was evaluated using real-time PCR. (C) Human synovial cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 or epiregulin in the absence of FBS for 3 hr. Ereg expression in the resulting cells was evaluated using real-time PCR. (D–F) Serum epiregulin concentrations in patients with (D) rheumatoid arthritis (n = 11), (E) atherosclerosis (n = 50), and (F) multiple sclerosis (n = 21) compared with healthy-aged, sex-matched subjects (rheumatoid arthritis, n = 26; atherosclerosis n = 64; multiple sclerosis, n = 15). Mean scores ± SD are shown. p values were calculated using Student’s t tests (* and #, p < 0.05; **p < 0.01; ***p < 0.001). Related to Figures S7, S8, and Table S10.

Article Snippet: Mouse Epiregulin ELISA Kit, Human soluble IL-6 receptor a, mouse IL-23, human epiregulin, mouse epiregulin, and anti-mouse epiregulin antibody were obtained from (R&D Systems).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Ophthalmology

Article Title: Epiregulin, epigen and betacellulin antibodies and axial elongation in young guinea pigs with lens-induced myopization

doi: 10.1186/s12886-022-02417-8

Figure Lengend Snippet: Axial length and axial elongation and vitreous cavity length and its elongation during the study period in young guinea pigs with bilateral lens-induced myopization and with unilateral intravitreal injections (right eyes) of epidermal growth factor family antibodies and contralateral intravitreal injections (left eyes) of phosphate buffered solution (mean ± standard deviations)

Article Snippet: In these three study groups, the animals received three intravitreal injections of antibodies against epiregulin (MAB1068, R&D Systems, Bio-Techne Co., Minnesota, USA), epigen (MAB11271, R&D Systems, Bio-Techne Co., Minnesota, USA), and betacellulin (AF1025, R&D Systems, Bio-Techne Co., Minnesota, USA), respectively, in a dose of 20 μg into their right eyes in weekly intervals.

Techniques:

Journal: BMC Ophthalmology

Article Title: Epiregulin, epigen and betacellulin antibodies and axial elongation in young guinea pigs with lens-induced myopization

doi: 10.1186/s12886-022-02417-8

Figure Lengend Snippet: Interocular difference in axial length and in axial elongation in young guinea pigs with bilateral lens-induced myopization and with unilateral intravitreal injections (right eyes) of epidermal growth factor family antibodies and contralateral intravitreal injections (left eyes) of phosphate buffered solution (mean ± standard deviations)

Article Snippet: In these three study groups, the animals received three intravitreal injections of antibodies against epiregulin (MAB1068, R&D Systems, Bio-Techne Co., Minnesota, USA), epigen (MAB11271, R&D Systems, Bio-Techne Co., Minnesota, USA), and betacellulin (AF1025, R&D Systems, Bio-Techne Co., Minnesota, USA), respectively, in a dose of 20 μg into their right eyes in weekly intervals.

Techniques: Significance Assay

Journal: BMC Ophthalmology

Article Title: Epiregulin, epigen and betacellulin antibodies and axial elongation in young guinea pigs with lens-induced myopization

doi: 10.1186/s12886-022-02417-8

Figure Lengend Snippet: Graph showing the distribution of the interocular difference (left eye minus right eye) in axial elongation during the study period at one week (blue bars), two weeks (green bars) and three weeks (red bars) after baseline in guinea pigs with bilateral lens-induced axial elongation and receiving intravitreal injections of antibodies to epiregulin, epigen and betacellulin

Article Snippet: In these three study groups, the animals received three intravitreal injections of antibodies against epiregulin (MAB1068, R&D Systems, Bio-Techne Co., Minnesota, USA), epigen (MAB11271, R&D Systems, Bio-Techne Co., Minnesota, USA), and betacellulin (AF1025, R&D Systems, Bio-Techne Co., Minnesota, USA), respectively, in a dose of 20 μg into their right eyes in weekly intervals.

Techniques:

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Sankey diagram of enriched receptor-ligand pairs in SSc skin and at least two lung scRNA-Seq datasets. Ribbon width is proportional to 1/rank of the skin SSc data. (B) Plot of the CellphoneDB ranks (adjusted p-values) of the interaction of EREG with EGFR in our skin scRNA-Seq data as well as our analysis of available data from SSc skin (15) and lung (33, 41, 42). Dotted line shows rank = 0.05. (C) Ereg relative expression during a time course of tissue digestion of healthy mouse skin, n=3 per time point. (D) Expression of EREG in our UMAP embedded scRNA-Seq data. (E) Heatmap of co-expressed genes by SSc EREG-expressing APC (EREG+) compared to healthy EREG+ APC and EREG− APC groups. For clarity, the raw gene list was filtered to genes primarily expressed by immune cells. (F) Expression of FCN1 in our UMAP embedded scRNA-Seq data. (G) Immunofluorescence images of EREG and FCN1 in SSc skin. (H, I) Analysis of EREG expression in SSc compared to healthy controls (H) and compared to modified Rodnan Skin Score (mRSS) (I) using data from (49). (J) Low and high magnification photomicrographs of skin and lung from SSc and healthy subject samples stained with EREG antibody. Dashed boxes delineate region shown in higher magnification image. Arrowheads label positive cells. (K) Enumeration of EREG+ cells in SSc skin dermis and lung (n=3 slides each, skin samples from patients SSc1, 3, and 4, 10 high power fields (hpf) per slide). Slides were imaged with a Keyence BZ-X800 microscope. Low power images are at 10x magnification and stitched together. High power images are 40x magnification. Data are means ± SD (***P<0.001, ****P<0.0001) analyzed with one-way analysis of variance (ANOVA) with Tukey multiple-comparisons test (C) and unpaired two-tailed Student t test (K).

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Expressing, Immunofluorescence, Modification, Staining, Microscopy, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Expression fold change of EREG when THP-1 monocytes were incubated with each indicated cytokine. (B) EREG protein quantification from supernatant of THP-1 incubated with IFNa2 for 4 hours, n=4 per group, data is representative from 2 independent experiments. (C-E) EREG expression fold change from freshly isolated peripheral blood CD14+ monocytes (C) and CD1c+ dendritic cell precursors (D) or cultured human BMDC (E) after incubation with IFNα2. (F) Expression fold change of NOTCH ligands, receptors, and target genes by HFFs incubated with recombinant human EREG (n=5). (G) HES1 expression fold change in SSc fibroblasts after incubation with EREG for 4 hours, n=5 per group. (H) EREG relative expression by BMDC primed with IFNα2 prior to exposure to NOTCH ligand DLL4 (n=3–4 per time point in each group). Statistics compare each group ± DLL4. (I) Relative expression of EGFR ligands by HFF (n=3). Genes with fewer than three points were below detectable level. (J) Changes in ECM gene expression when HFF were incubated with media alone (NT) or EREG neutralizing antibody (Ereg Ab). FNEDA refers to the extra domain A-containing isoform of fibronectin (n=5 per group). (K) Model of EREG-NOTCH circuit between monocyte-derived DC3 and fibroblasts. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001) analyzed with unpaired two-tailed Student t test (A-H, J) and one-way ANOVA with Tukey multiple-comparisons test (I).

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Expressing, Incubation, Isolation, Cell Culture, Recombinant, Gene Expression, Derivative Assay, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Experimental diagram depicting adjacent punch biopsies obtained from the forearm of a patient with diffuse cutaneous SSc, which were cultured for 9 days in media alone (NT) or with addition of EREG neutralizing antibody (Ereg Ab). (B) Histology of cultured skin explants, with inset showing higher magnification of dermal collagen. (C, D) Skin explant media was analyzed for pro-COL1A1 N-terminal peptide (PINP) and TNC. (E) Percent reduction of protein by Ereg Ab treatment compared to NT control. (F) LDH activity of skin explant supernatants from patient SSc7. (G-N) Fresh explanted lung tissue from a deceased patient donor with familial idiopathic pulmonary fibrosis was processed for histologic staining, which showed fibroblastic foci formation and hyperplasia of alveolar type II epithelial cells, indicated by arrows (left panel H&E, right panel trichrome). The same tissue was cut into cubes and cultured for 10 days in the presence of the multikinase inhibitor nintedanib (Nin), the Alk5 inhibitor A-1544033 (IN-1130) (Alk5i), EREG antibody (Ereg Ab) or non-treated vehicle control (NT). (H-K) Relative expression of indicated genes, n=4 per group. (L-N) Protein secretion of indicated genes measured by ELISA, n=8 per group. ELISA samples with poor signal and qPCR outliers identified by Grubbs’s test with alpha = 0.05 were excluded. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ****P < 0.0001) analyzed with paired two-tailed Student t test (C-E) comparing NT and Ereg Ab treated samples. In (H-N), comparison of each inhibitor to NT control was analyzed by one-way ANOVA with Dunnett’s multiple-comparisons test whereas Ereg Ab was individually compared to Nin and Alk5i by unpaired two-tailed Student t test.

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Cell Culture, Control, Activity Assay, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A-C) B6 mice were injected subcutaneously with 0.2 mg bleomycin (BLM) and 3 weeks later skin was stained for hematoxylin and eosin (A) and trichrome (B). Epidermis (epi), dermis (dermis) and dermal white adipose tissue (DWAT) are highlighted on histology. (C, D) Immunofluorescence images of PBS and BLM-treated skin 3 weeks post-injection. (E) Hydroxyproline content of the skin at different time points after subcutaneous bleomycin injection (n=3 per group). (F) Heatmap of mean log2(expression fold change) of ECM genes and EGFR ligands at different time points after subcutaneous bleomycin injection compared to the mean of each group and PBS controls, n=3 per time point. (G) Bulk RNA sequencing of dendritic cells isolated from fibrotic skin of Mgl2DTReGFPpANeo mice 3 weeks after subcutaneous bleomycin injection compared to PBS controls (n=3 per group). (H) Relative expression of Ereg at different time points after intratracheal bleomycin administration to B6 mice. (I) B6 mice were injected with bleomycin subcutaneously, then at 2 weeks injected intraperitoneally with Ifnar1-blocking antibody (Ifnar1 Ab), isotype control antibody (iso) or not treated (NT). No significant differences were found between NT and isotype Ab control groups, so they were combined for clarity. At 3 weeks, skin was analyzed for histology (J), dermal skin thickness (K), hydroxyproline (L), and gene expression (M, N). Data from E and F, G, and H are single independent experiments. Data in J-N are aggregated from two separate experiments. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001) analyzed with one-way ANOVA with Tukey multiple-comparisons test (E, F, H, K) and unpaired two-tailed Student t test (L-N).

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Injection, Staining, Immunofluorescence, Expressing, RNA Sequencing, Isolation, Blocking Assay, Control, Gene Expression, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A-C) Diagramed in (A), cohorts of B6 and Ereg−/− mice were injected with bleomycin subcutaneously and 35 days later analyzed for skin thickness (B) and histology (C). (D-I) Diagramed in (D), 21 days after bleomycin injection mice began treatment with Ereg antibody (Ereg Ab) compared to controls treated with PBS (NT) for two weeks. Skin was analyzed for dermal thickness (E), hydroxyproline (F), gene expression (G, H) and histology (I), with H&E staining on the top row, trichrome in the middle row, and pEGFR immunohistochemistry (IHC) on the bottom row, n=8 (PBS), 11 (BLM), and 12 (Ereg Ab). (J) B6 and Ereg−/− mice were injected with bleomycin or PBS and 3 weeks later B6 mice were treated for 1 week with Ereg Ab or isotype control Ab, n=3 (PBS), 5 (isotype Ab), 5 (Ereg Ab), and 5 NT Ereg−/−. (K-L) As diagramed in (K), 10 days after intratracheal bleomycin mice were treated with Ereg Ab for two weeks. Lungs were analyzed for histology (L), modified Ashcroft score (M), hydroxyproline (N) and Ereg gene expression (O), n=6 (PBS), 11 (BLM), and 7 (Ereg Ab). Histology images of skin and lung are 10x and 20x magnification, respectively. IHC images are 40x magnification. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P<0.001, ****P<0.0001) analyzed with unpaired two-tailed Student t test (F-H, N, O) and one-way ANOVA with Tukey multiple-comparisons test (B, E, J, M). Data for J is single experiment and data for A-C, D-I, and K-O are aggregated from two independent experiments.

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Injection, Gene Expression, Staining, Immunohistochemistry, Control, Modification, Two Tailed Test

Journal: Pain

Article Title: The dichotomous role of epiregulin in pain

doi: 10.1097/j.pain.0000000000001792

Figure Lengend Snippet: Quantile-quantile plot of 2,407 SNPs in genes coding for EGFR superfamily receptors and ligands, showing a significant association (FDR < 0.05) between seven EREG SNPs and chronic characteristic pain intensity (CPI) in OPPERA cohort.

Article Snippet: Anti-EREG monoclonal antibody (mAb) A blocking/neutralizing EREG monoclonal antibody (mAb) (NBP2–21992, Novus Biologicals, Oakville, ON) was diluted in phosphate buffered saline (PBS) and administered directly into the tail vein (5 μg/5 μl).

Techniques:

Journal: Pain

Article Title: The dichotomous role of epiregulin in pain

doi: 10.1097/j.pain.0000000000001792

Figure Lengend Snippet: Linear regression analyses of all the genes in EGFR receptor family and its ligands, and, chronic characteristic pain intensity (CPI) in OPPERA cohort, corrected for age, gender and the first three principal components. Only significant results (FDR ≤ 5%) are presented.

Article Snippet: Anti-EREG monoclonal antibody (mAb) A blocking/neutralizing EREG monoclonal antibody (mAb) (NBP2–21992, Novus Biologicals, Oakville, ON) was diluted in phosphate buffered saline (PBS) and administered directly into the tail vein (5 μg/5 μl).

Techniques:

Journal: Pain

Article Title: The dichotomous role of epiregulin in pain

doi: 10.1097/j.pain.0000000000001792

Figure Lengend Snippet: The EREG gene has two minor haplotypes. (A) Regional plot of EREG. (B) Illustration of the 16 SNPs in EREG Linkage Disequilibrium plot, numbers inside each cell indicate r2 values, color reflects D’ value, ranging from white to red, (i.e. 0 to 1). (C) The sequence of three haplotypes with frequency > 5% within EREG gene locus. Major and minor alleles of EREG SNPs genotyped in OPPERA, SNPs significantly associated with CPI from Figure 2 are highlighted in green. Marker SNPs, namely, rs1993665, rs2367707, and rs6836436, for haplotypes H1, H2, and H3, respectively, are in highlighted in yellow.

Article Snippet: Anti-EREG monoclonal antibody (mAb) A blocking/neutralizing EREG monoclonal antibody (mAb) (NBP2–21992, Novus Biologicals, Oakville, ON) was diluted in phosphate buffered saline (PBS) and administered directly into the tail vein (5 μg/5 μl).

Techniques: Sequencing, Marker

Journal: Pain

Article Title: The dichotomous role of epiregulin in pain

doi: 10.1097/j.pain.0000000000001792

Figure Lengend Snippet: Haplotype frequencies of EREG as estimated through Expectation – Maximization (E-M) Algorithm:

Article Snippet: Anti-EREG monoclonal antibody (mAb) A blocking/neutralizing EREG monoclonal antibody (mAb) (NBP2–21992, Novus Biologicals, Oakville, ON) was diluted in phosphate buffered saline (PBS) and administered directly into the tail vein (5 μg/5 μl).

Techniques:

Journal: Pain

Article Title: The dichotomous role of epiregulin in pain

doi: 10.1097/j.pain.0000000000001792

Figure Lengend Snippet: cis – eQTL in blood for the marker SNPs of minor haplotypes in EREG :

Article Snippet: Anti-EREG monoclonal antibody (mAb) A blocking/neutralizing EREG monoclonal antibody (mAb) (NBP2–21992, Novus Biologicals, Oakville, ON) was diluted in phosphate buffered saline (PBS) and administered directly into the tail vein (5 μg/5 μl).

Techniques: Marker

Journal: Pain

Article Title: The dichotomous role of epiregulin in pain

doi: 10.1097/j.pain.0000000000001792

Figure Lengend Snippet: H3 and H2 haplotypes of EREG protects from chronic clinical pain. (A-B) OPPERA cohort. (A) Bar plot of average minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) among chronic TMD cases and controls and (B) Plot of mean chronic pain intensity at baseline for minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) in the OPPERA cohort. (C-D) UKB cohort. (C) Bar plot of average minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) among chronic pain cases (at least one chronic pain site) and controls and (D) Plot of mean number of chronic pain sites for minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) in the UKB cohort. Symbols represent mean ± SEM; False Discovery Rates (FDR) were derived by generalized linear modelling for haplotype association; *FDR < 0.05; **FDR < 0.01.

Article Snippet: Anti-EREG monoclonal antibody (mAb) A blocking/neutralizing EREG monoclonal antibody (mAb) (NBP2–21992, Novus Biologicals, Oakville, ON) was diluted in phosphate buffered saline (PBS) and administered directly into the tail vein (5 μg/5 μl).

Techniques: Derivative Assay

Journal: Pain

Article Title: The dichotomous role of epiregulin in pain

doi: 10.1097/j.pain.0000000000001792

Figure Lengend Snippet: Haplotype association to analyze the relationship between chronic pain phenotypes and EREG haplotypes:

Article Snippet: Anti-EREG monoclonal antibody (mAb) A blocking/neutralizing EREG monoclonal antibody (mAb) (NBP2–21992, Novus Biologicals, Oakville, ON) was diluted in phosphate buffered saline (PBS) and administered directly into the tail vein (5 μg/5 μl).

Techniques:

Journal: Pain

Article Title: The dichotomous role of epiregulin in pain

doi: 10.1097/j.pain.0000000000001792

Figure Lengend Snippet: H3 haplotype of EREG is a risk for acute clinical pain. (A-B) OPPERA cohort. (A) Bar plot of average minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) among acute facial pain cases and controls and (B) Plot of mean of acute pain intensity at follow-up in controls for minor allele counts of rs1993665, rs2367707 and rs6836436, (markers for haplotypes H1, H2 and H3, respectively) in the OPPERA cohort. (C-D) UKB cohort. (C) Bar plot of average minor allele counts of rs1993665, rs2367707, and rs6836436, (markers for haplotypes H1, H2, and H3, respectively) among acute pain cases (at least one acute pain site) and controls. (D) A plot of the mean number of acute pain sites for minor allele counts of rs1993665, rs2367707, and rs6836436, (markers for haplotypes H1, H2, and H3, respectively) in the UK biobank (UKB) cohort. Symbols represent mean ± SEM; False Discovery Rates (FDR) were derived by generalized linear modelling for haplotype association; *FDR < 0.05; **FDR < 0.01.

Article Snippet: Anti-EREG monoclonal antibody (mAb) A blocking/neutralizing EREG monoclonal antibody (mAb) (NBP2–21992, Novus Biologicals, Oakville, ON) was diluted in phosphate buffered saline (PBS) and administered directly into the tail vein (5 μg/5 μl).

Techniques: Derivative Assay

Journal: Pain

Article Title: The dichotomous role of epiregulin in pain

doi: 10.1097/j.pain.0000000000001792

Figure Lengend Snippet: Haplotype association to analyze the relationship between acute pain phenotypes and EREG haplotypes:

Article Snippet: Anti-EREG monoclonal antibody (mAb) A blocking/neutralizing EREG monoclonal antibody (mAb) (NBP2–21992, Novus Biologicals, Oakville, ON) was diluted in phosphate buffered saline (PBS) and administered directly into the tail vein (5 μg/5 μl).

Techniques:

Journal: Pain

Article Title: The dichotomous role of epiregulin in pain

doi: 10.1097/j.pain.0000000000001792

Figure Lengend Snippet: The effects of systemically administering an EREG monoclonal antibody (mAb, 5 μg) in mouse models of pain. (A) Mice injected with the EREG mAb 3 days following CFA, as indicated by the arrow have higher paw withdrawal thresholds (g/mm2) compared with control mice 5 days post-CFA; n = 8/group. (B) The EREG mAb or vehicle control was administered 14 days following SNI surgery, as indicated by the arrow. A single administration of the mAb reverses mechanical allodynia for up to one week. (C) The concentration of EREG mAb in the blood plasma of mice following a single tail vein administration; n = 4/group. (D) Mice injected with the EREG mAb 1 day following CFA, as indicated by the arrow have lower paw withdrawal thresholds (g/mm2) than control mice 7 days post-CFA; n = 8/group. (E) Pre-treatment with the EREG mAb, 2 days before testing increases nocifensive behavior in the intraplantar capsaicin test of acute pain; n = 18–20/group. (F) A subset of mice from E were tested for mechanosensitivity following intraplantar capsaicin injection; n = 8/group. Mice injected with the EREG mAb have lower paw withdrawal thresholds (g/mm2) in the capsaicin injected paw, but not the uninjected paw when compared with controls. BL: baseline.*p < 0.05; **p < 0.001; ***p < 0.001 compared with vehicle at the indicated time points. †p < 0.05; ††p < 0.001 compared with capsaicin injected paw in F.

Article Snippet: Anti-EREG monoclonal antibody (mAb) A blocking/neutralizing EREG monoclonal antibody (mAb) (NBP2–21992, Novus Biologicals, Oakville, ON) was diluted in phosphate buffered saline (PBS) and administered directly into the tail vein (5 μg/5 μl).

Techniques: Injection, Control, Concentration Assay, Clinical Proteomics