Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG improved glucose tolerance in the absence of leptin in Lep ob mice and exhibited no effect in LepR-deficient Lepr db mice. ( A ) Body weight of Lep ob male mice in groups before and after treatment with Veh (PBS, white bar) or EREG (50 ng/g body weight (BW), black bar) for 26 days. Mice were on regular chow diet. Unpaired t -test, n = 7/group. ns, not significant. ( B , C ) Fat ( B ) and lean body ( C ) composition in same groups of mice at the end of the study was measured by Echo-MRI. Fat and lean mass are shown as % of the total weight (100%). ( D , E ) Glucose tolerance test (GTT) was performed in fasted Lep ob mice treated with PBS (Veh, open circles) or EREG (closed circles) ( n = 7 per group). GTT kinetics ( D ) and area under the curve (AUC) ( E ) are shown. Statistical significance was examined by ANOVA ( D ) and Student’s t -test ( E ). ( F ) Insulin levels in plasma in both mouse groups were measured by ELISA. Unpaired student’s t -test. ( G ) Weight before and after treatment of Lepr db male mice with Veh (PBS, white bar) or EREG (50 ng/g body weight (BW), black bar) for 4 weeks ( n = 6 per treatment). Mice were on regular chow. Unpaired Student’s t -test, n = 6/group. ( H , I ) Fat ( H ) and lean body ( I ) composition (% of total weight) in the same groups of mice at the end of the study were measured by Echo-MRI. ( J , K ) GTT kinetics ( J ) and AUC ( K ) were obtained from Lepr db mice treated with PBS (Veh, open circles) or EREG (closed circles). ANOVA ( J ) and Student’s t -test ( K ). ( L ) Insulin levels in plasma in both mouse groups were measured by ELISA. Unpaired student’s t -test.

Article Snippet: Mouse recombinant EREG (50599-M01H, Sino Biological Beijing, China) or Creative Biomart (No. Ereg-576M, New York, NY, USA) and human recombinant EREG (1195-EP/CF, R&D Systems, Minneapolis, MN, USA) were used for in vitro assays and/or in vivo studies.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG regulated glucose uptake via binding with LepR in Lep ob mice. ( A ) EREG and insulin tolerance test in Lep ob mice ( n = 5 per group) treated with a single intraperitoneal injection of insulin (0.012 IU/g BW, triangle dashed line) or EREG (80 ng/g BW, closed circles. Asterisks, significant (* p < 0.05) compared to glucose levels before EREG treatment. # Hashtag, significant difference in glucose levels 30 min after treatment with EREG or insulin. Unpaired Student’s t -test. ( B ) Area under the curve (AUC) quantification of insulin (hatched bar) and EREG (black bar) tolerance tests. Unpaired Student’s t -test, ns . ( C ) GTT kinetics were measured in Lep ob mice ( n = 5 per treatment) treated with a single injection of PBS (Veh, open circles) or EREG (closed circles). Student’s t -test. * p < 0.05 from comparison between control and EREG treated mice at each time point. ( D ) Area under the curve (AUC) quantification of insulin (hatched bar) and EREG (black bar) tolerance tests. Unpaired Student’s t -test. ( E , F ). Immunoprecipitation of LepR was performed with anti-EREG antibody using homogenates from subcutaneous fat ( C ) and visceral fat ( D ). Fat tissue was isolated from non-treated Lep ob (Veh) as well as Lep ob mice 15 min after injection of EREG (50 ng/mL).

Article Snippet: Mouse recombinant EREG (50599-M01H, Sino Biological Beijing, China) or Creative Biomart (No. Ereg-576M, New York, NY, USA) and human recombinant EREG (1195-EP/CF, R&D Systems, Minneapolis, MN, USA) were used for in vitro assays and/or in vivo studies.

Techniques: Binding Assay, Injection, Comparison, Control, Immunoprecipitation, Isolation

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG-stimulated glucose uptake was dependent on LepR but independent of EGFR. ( A , B ) Fluorescently-labelled (FD) glucose uptake was measured in stromal vascular fraction (SVF) cells isolated from visceral tissues of Lepr db ( A ) or Lep ob mice ( B ). Cells were treated with either Veh (PBS), mouse EREG (50 ng/mL), human insulin (Ins, 10 µg/mL), or mouse leptin (Lep, 200 ng/mL) for 80 min. For inhibition experiment, Lep ob SVF cells were pre-treated with EGFR inhibitor (EGFR-I, AST-1306, 10 µM) or vehicle (Veh, DMSO) for 40 min. Data are shown as a percentage of Veh-treated control (100%, n = 8 per treatment). Unpaired Student’s t -test. ( C – E ) FD-glucose uptake was measured in mouse 3T3-L3 preadipocytes. ( C ) Preadipocytes were treated with vehicle, human insulin (Ins, 10 µg/mL), and mouse EREG (50 ng/mL) for 30 min (mean ± SEM, n = 6, t -test). ( D ) Time-dependent uptake of FD-glucose in 3T3-L1 preadipocytes stimulated with human insulin (Ins, 10 µg/mL), mouse leptin (Lep, 200 ng/mL), and mouse EREG (50 ng/mL). Data are shown (mean ± SEM, n = 8, t -test) as % of glucose uptake compared to control cells at the same time point (Veh, 100%). ( E ) Concentration-dependent increase in FD-glucose uptake by 3T3-L1 preadipocytes stimulated with different concentrations of mouse EREG. Data are shown as a percentage of Veh-treated control (100%, n = 6 per concentration). * p < 0.05, significant differences compared to the vehicle group, one-way ANOVA). ( F ) NIH-3T3 preadipocytes were transiently transfected with pB- Glut4 -7myc-GFP and stimulated with vehicle, Ins (10 µg/mL), EREG (50 ng/mL) for 60 min. Data show representative fluorescent images of GFP-labeled GLUT4 selected from three independent experiments. 10× magnification. Yellow arrow shows GFP-labeled GLUT4 that was translocated to the cellular membrane. ( G ) Quantification of GFP was performed in adipocytes of similar size ( n = 10) in each group.

Article Snippet: Mouse recombinant EREG (50599-M01H, Sino Biological Beijing, China) or Creative Biomart (No. Ereg-576M, New York, NY, USA) and human recombinant EREG (1195-EP/CF, R&D Systems, Minneapolis, MN, USA) were used for in vitro assays and/or in vivo studies.

Techniques: Isolation, Inhibition, Control, Concentration Assay, Transfection, Labeling, Membrane

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG mediates glucose uptake via PI3K with transient activation of ERK. ( A ) FD-glucose uptake in 3T3-L3 preadipocytes treated with or without EREG (50 ng/mL) and in the presence of inhibitors for EGFR-I (AG1478, 10 µM), EGFR and ErbB2 (AST-1306 or CI-1033 10 µM), dual IR/IGF-1R inhibitor (BMS 536924, 1 µM), and SRC-I, AZM475271, 1 µM) for 30 min. Cells were starved for 90 min before stimulation. Dashed line shows glucose uptake in the presence of insulin (Ins, 10 µg/mL). ( B ) FD-glucose uptake was measured in mouse 3T3-L1 preadipocytes with or without EREG (50 ng/mL) and inhibitors of MEK1/2 and PI3K (MEK1/2-I, U0126 10 μM, and PI3K-I, wortmannin 200 nM). Data (mean ± SD, n = 6) are shown as a percentage of control (Veh 100%). Unpaired Student’s t -test. ( C ) 3T3-L1 preadipocytes were stimulated with EREG at different concentrations (0–100 ng/mL) for 5 or 15 min. The total and phosphorylated levels of AKT, STAT3, STAT5, and ERK were measured by Western blot in duplicates. Data are shown in a representative Western blot. ( D ) The kinetics of pERK expression was quantified based on the Western blots. pAKT, p-STAT3, and p-STAT5 analysis are described in . Pearson correlation analysis. ( E ) 3T3-L1 preadipocytes were stimulated with or without EREG or EGF (50 ng/mL, each) for 30 min in the presence and absence of EGFR inhibitor AST1306 (100 nM), and antibody against mouse LepR (Invitrogen, PA1-053, 10 μg/mL). For inhibition, cells were pre-treated 30 min before EREG and EGF stimulation. ( F ) FD-glucose uptake was measured in mouse 3T3-L3 preadipocytes pre-treated with either Veh (DMSO) or ERK inhibitors (U0126, SCH772984, or DEL 22379, each 10 µM in DMSO) for 40 min. Then, cells were treated with either Veh (PBS), mouse EREG (50 ng/mL), or mouse leptin (Lep, 200 ng/mL) for 80 min. Data are shown as a percentage of Veh-treated control (100%, n = 7 per group). Unpaired Student’s t -test. ns , not significant ( p > 0.05).

Article Snippet: Mouse recombinant EREG (50599-M01H, Sino Biological Beijing, China) or Creative Biomart (No. Ereg-576M, New York, NY, USA) and human recombinant EREG (1195-EP/CF, R&D Systems, Minneapolis, MN, USA) were used for in vitro assays and/or in vivo studies.

Techniques: Activation Assay, Control, Western Blot, Expressing, Inhibition

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: Kinetics of the changes in LepR film thickness in the presence of leptin ( A ) or EREG ( B ). Film thickness was measured using QCM and quantified based on the binding kinetics to a gold sensor.

Article Snippet: Mouse recombinant EREG (50599-M01H, Sino Biological Beijing, China) or Creative Biomart (No. Ereg-576M, New York, NY, USA) and human recombinant EREG (1195-EP/CF, R&D Systems, Minneapolis, MN, USA) were used for in vitro assays and/or in vivo studies.

Techniques: Binding Assay

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: Evolutionary analysis of EREG binding to LepR. ( A – E ) EREG docking to LepR. Evolutionary analysis of 175 open The dependence of EREG-mediated glucose uptake on the ERK phosphorylation cascade was examined using (1) a specific inhibitor of ERK1/2 SCH772984 , (2) an inhibitor of ERK dimerization DEL-22379 , and (3) a selective inhibitor of MEK1 and MEK2 U0126 . All inhibitors increased basal glucose uptake, which was further increased by leptin ( F). The inhibition of ERK1/2 and MEK1/2 as well as ERK dimerization prevented stimulatory effect of EREG on FD-glucose uptake but did not decrease it beyond the levels seen in the control cells. Although transient ERK phosphorylation occurred in response to EREG stimulation, this pathway was dispensable for glucose uptake and dependent on PI3K and may be other pathways ( B and ). ( F ) Hypothetic mechanism suggesting EREG as an alternative ligand for both EGFR and LepR. The canonic leptin/LepR response can induce JAK/STAT3 signaling and required the long form of LepR. The alternative binding of EREG to LepR can induce ERK and PI3K activation increasing GLUT4 translocation and glucose uptake, but not the other canonic effects of leptin, including the regulation of appetite and energy expenditure.

Article Snippet: Mouse recombinant EREG (50599-M01H, Sino Biological Beijing, China) or Creative Biomart (No. Ereg-576M, New York, NY, USA) and human recombinant EREG (1195-EP/CF, R&D Systems, Minneapolis, MN, USA) were used for in vitro assays and/or in vivo studies.

Techniques: Binding Assay, Phospho-proteomics, Inhibition, Control, Activation Assay, Translocation Assay

Journal: bioRxiv

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

doi: 10.64898/2026.04.07.717082

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

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

Techniques: Staining, Immunohistochemical staining, Expressing, Immunostaining

Journal: bioRxiv

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

doi: 10.64898/2026.04.07.717082

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

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

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

Journal: Cell structure and function

Article Title: Potential involvement of Twist2 and Erk in the regulation of osteoblastogenesis by HB-EGF-EGFR signaling.

doi: 10.1247/csf.10001

Figure Lengend Snippet: Fig. 1. EGFR signaling suppresses osteoblast differentiation through Ras. A) Expression of EGF family members mRNA in osteoblasts. MC3T3-E1 cells were induced to differentiate into osteoblasts, and the mRNA expression of EGF family members was monitored by RT-PCR at the indicated time points. GAPDH was used as an internal control. Hb-egf, heparin-biding EGF-like growth factor; Tgfα, transforming growth factor-α; Areg, amphiregulin; Epr, epiregulin; Btc, betacellulin. P, positive control. mRNA from mouse heart, liver, and lung was used as a positive control. B) Effect of rHB-EGF on osteoblastogenesis. MC3T3-E1 cells were seeded on 24-well plates, and osteoblast differentiation was induced. Alkaline phosphatase (ALP) activity and mineralizing activity were measured by ALP staining (upper panel) and Alizarin red staining (lower panel), respectively, at the indicated time points. C) Expression of osteoblastic marker genes in MC3T3-E1 cells. Cells were cultured in osteogenic media in the presence of rHB-EGF, and the mRNA expression of the indicated genes was monitored by RT-PCR. ColI, Collagen type I; Alp, alkaline phosphatase; Opn, osteopontin; Ocn, osteocalcin. GAPDH was used as an internal control. D) Activation of ERK in osteoblast differentiation. The phosphorylated form of ERK was visualized by Western blotting. MC3T3-E1 cells were stimulated with rHB-EGF for 5 min in the presence of the indicated inhibitors. A.A., ascorbic acid; HB, rHB-EGF; αHB, anti-human HB-EGF neutralizing antibody; PD, PD98059, MEK inhibitor; AG, AG1478, EGFR inhibitor. E) MC3T3-E1 cells were cultured in osteogenic media stimulated with rHB-EGF and indicated inhibitors as shown in D) for 3 days and ALP staining was carried out. F) Involvement of Ras in osteoblast differentiation. A dominant-negative form of Ras was expressed in MC3T3-E1 cells, and ALP staining was performed three days after osteogenic stimulation in the presence or absence of rHB-EGF. Mock, empty vector.

Article Snippet: Recombinant human HB-EGF protein (rHB-EGF), recombinant human epiregulin protein (rEpr) and anti-human HB-EGF antibody were purchased from R&D Systems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Positive Control, Activity Assay, Staining, Marker, Cell Culture, Activation Assay, Western Blot, Dominant Negative Mutation, Plasmid Preparation

Journal: The Journal of clinical endocrinology and metabolism

Article Title: TGF-β1 induces COX-2 expression and PGE2 production in human granulosa cells through Smad signaling pathways.

doi: 10.1210/jc.2013-4100

Figure Lengend Snippet: Figure 3. TGF-1 enhances AREG-, BTC-, and EREG-induced COX-2 expression in SVOG cells. A, SVOG cells were treated with 50 ng/mL AREG, BTC, or EREG alone or in combination with 5 ng/mL TGF-1 for 1 hour. The mRNA levels of COX-2 were examined by RT-qPCR. Ctrl, control. B, SVOG cells were treated with 50 ng/mL AREG, BTC, or EREG alone or in combination with 5 ng/mL TGF-1 for 3 hours. The protein levels of COX-2 were examined by Western blotting. The results are expressed as the means SEM of at least three independent experiments. Values without a common letter were significantly different (P .05).

Article Snippet: Recombinant human TGF- 1, amphiregulin, betacellulin, and epiregulin were obtained from R&D Systems.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: The Journal of clinical endocrinology and metabolism

Article Title: TGF-β1 induces COX-2 expression and PGE2 production in human granulosa cells through Smad signaling pathways.

doi: 10.1210/jc.2013-4100

Figure Lengend Snippet: Figure 6. TGF-1 induces PGE2 production in SVOG cells. A–D, SVOG cells were treated with 5 ng/mL TGF-1 for 3 and 6 hours (A). SVOG cells were treated with 50 ng/mL AREG, BTC, or EREG alone or in combination with 5 ng/mL TGF-1 for 3 hours (B). SVOG cells were treated with 5 ng/mL TGF-1 in combination with SB431542 (10 M) for 6 hours (C). SVOG cells were transfected with a 50-nM control siRNA (si-Ctrl), a Smad2 siRNA (si-Smad2), or a Smad3 siRNA (si-Smad3) for 48 hours and then treated with 5 ng/mL TGF-1 for 6 hours (D). The levels of PGE2 in culture media were examined by ELISA. Ctrl, control. E, SVOG cells were transfected with a 50-nM control siRNA (si-Ctrl) or a COX-2 siRNA (si-COX-2) for 48 hours and then treated with 5 ng/mL TGF-1 for 3 hours. The protein levels of COX-2 were examined by Western blotting. F, SVOG cells were transfected with a 50-nM control siRNA (si-Ctrl) or a COX-2 siRNA (si-COX-2) for 48 hours and then treated with 5 ng/mL TGF-1 for 6 hours. The levels of PGE2 in culture media were examined by ELISA. The results are expressed as the means SEM of at least three independent experiments. Values without a common letter were significantly different (P .05).

Article Snippet: Recombinant human TGF- 1, amphiregulin, betacellulin, and epiregulin were obtained from R&D Systems.

Techniques: Transfection, Control, Enzyme-linked Immunosorbent Assay, Western Blot