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: The Journal of clinical endocrinology and metabolism

Article Title: EGF-like growth factors induce COX-2-derived PGE2 production through ERK1/2 in human granulosa cells.

doi: 10.1210/jc.2013-2662

Figure Lengend Snippet: Figure 1. LH upregulates AREG, BTC, and EREG mRNA levels in SVOG cells. A, SVOG cells were treated with 100 ng/mL LH, and the mRNA levels of AREG, BTC, and EREG were analyzed at different time points using RT-qPCR. B, SVOG cells were treated with 1mM 8-Br-cAMP, and the mRNA levels of AREG, BTC, and EREG were analyzed at different time points using RT-qPCR. RT- qPCR results are expressed as the mean SEM of at least 3 independent experiments. Values without a common letter were significantly different (P .05). Abbreviation: Ctrl, control.

Article Snippet: Recombinant human AREG, BTC, and EREG were obtained from R&D Systems.

Techniques: Quantitative RT-PCR, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: EGF-like growth factors induce COX-2-derived PGE2 production through ERK1/2 in human granulosa cells.

doi: 10.1210/jc.2013-2662

Figure Lengend Snippet: Figure 3. AREG, BTC, and EREG upregulate COX-2 expression in SVOG cells. A, SVOG cells were treated with 50 ng/mL AREG, BTC, or EREG, and the mRNA levels of COX-1 (upper panel) and COX-2 (lower panel) were analyzed at different time points using RT-qPCR. B, SVOG cells were treated with increasing concentrations of AREG, BTC, or EREG (1, 10, 20, 50, and 100 ng/mL) for 1 hour, and the mRNA levels of COX-1 (upper panel) and COX-2 (lower panel) were examined using RT-qPCR. RT-qPCR results are expressed as the mean SEM of at least 3 independent experiments. C and D, SVOG cells were treated with 50 ng/mL AREG, BTC, or EREG for 1 and 3 hours. The protein levels of COX-1 (C) and COX-2 (D) were analyzed using Western blotting. The top panel presents representative results of the Western blotting analyses. The bottom panel presents summarized quantitative results that are expressed as the mean SEM of at least 3 independent experiments. Values without a common letter were significantly different (P .05). Abbreviation: Ctrl, control.

Article Snippet: Recombinant human AREG, BTC, and EREG were obtained from R&D Systems.

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

Journal: The Journal of clinical endocrinology and metabolism

Article Title: EGF-like growth factors induce COX-2-derived PGE2 production through ERK1/2 in human granulosa cells.

doi: 10.1210/jc.2013-2662

Figure Lengend Snippet: Figure 4. EGFR is required for AREG-, BTC-, and EREG-induced upregulation of COX-2 expression. A and B, SVOG cells were pretreated with vehicle control (dimethylsulfoxide) or 10M AG1478 for 30 minutes, followed by treatment with 50 ng/mL AREG, BTC, or EREG for 1 hour. The COX-2 mRNA (A) and protein (B) levels were examined by RT-qPCR and Western blotting, respectively. C and D, SVOG cells were transfected with 50nM control siRNA or EGFR siRNA for 48 hours and were treated with 50 ng/mL AREG, BTC, or EREG for 1 hour. The EGFR and COX-2 mRNA (C) and protein (D) levels were examined by RT-qPCR and Western blotting, respectively. Results are expressed as the mean SEM of at least 3 independent experiments. Values without a common letter were significantly different (P .05). Abbreviations: Ctrl, control; DMSO, dimethylsulfoxide.

Article Snippet: Recombinant human AREG, BTC, and EREG were obtained from R&D Systems.

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

Journal: The Journal of clinical endocrinology and metabolism

Article Title: EGF-like growth factors induce COX-2-derived PGE2 production through ERK1/2 in human granulosa cells.

doi: 10.1210/jc.2013-2662

Figure Lengend Snippet: Figure 5. ERK1/2 activation is required for AREG-, BTC-, and EREG-induced upregulation of COX-2 expression. A, SVOG cells were treated with 50 ng/mL AREG, BTC, or EREG for 10 and 30 minutes. Phosphorylation of ERK1/2 and Akt were determined by Western blotting using antibodies specific for phosphorylated, activated forms of ERK1/2 (p-ERK1/2) and Akt (p-Akt). Membranes were stripped and reprobed with antibodies to total ERK1/2 and Akt. B, SVOG cells were pretreated with vehicle control (dimethylsulfoxide), 10M U0126, or 10M LY294002 for 30 minutes and then treated with 50 ng/mL AREG, BTC, or EREG for 1 hour. The COX-2 mRNA levels were examined by RT-qPCR. C, SVOG cells were pretreated with vehicle control (dimethylsulfoxide) or 10M U0126 for 30 minutes and then treated with 50 ng/mL AREG, BTC, or EREG for 1 hour. The COX-2 protein levels were examined by Western blotting. Results are expressed as the mean SEM of at least 3 independent experiments. Values without a common letter were significantly different (P .05). Abbreviations: Ctrl, control; DMSO, dimethylsulfoxide.

Article Snippet: Recombinant human AREG, BTC, and EREG were obtained from R&D Systems.

Techniques: Activation Assay, Expressing, Phospho-proteomics, Western Blot, Control, Quantitative RT-PCR

Journal: The Journal of clinical endocrinology and metabolism

Article Title: EGF-like growth factors induce COX-2-derived PGE2 production through ERK1/2 in human granulosa cells.

doi: 10.1210/jc.2013-2662

Figure Lengend Snippet: Figure 6. AREG, BTC, and EREG induce PGE2 production in SVOG cells. A, SVOG cells were treated with 50 ng/mL AREG, BTC, or EREG for 3 hours. The levels of PGE2 in culture media were examined by ELISA. B, SVOG cells were transfected with 50nM control siRNA or COX-2 siRNA for 48 hours and were treated with 50 ng/mL AREG, BTC, or EREG for 3 hours. The protein levels of COX-2 were analyzed using Western blotting. C, SVOG cells were transfected with 50nM control siRNA or COX-2 siRNA for 48 hours and were treated with 50 ng/mL AREG, BTC, or EREG for 3 hours. The levels of PGE2 in culture media were examined by ELISA. D, SVOG cells were pretreated with vehicle control (dimethylsulfoxide) or 10M AG1478 for 30 minutes and then treated with 50 ng/mL AREG, BTC, or EREG for 3 hours. The levels of PGE2 in culture media were examined by ELISA. E, SVOG cells were pretreated with vehicle control (dimethylsulfoxide) or 10M U0126 for 30 minutes and then treated with 50 ng/mL AREG, BTC, or EREG for 3 hours. The levels of PGE2 in culture media were examined by ELISA. Results are expressed as the mean SEM of at least 3 independent experiments. Values without a common letter were significantly different (P .05). Abbreviations: Ctrl, control; DMSO, dimethylsulfoxide.

Article Snippet: Recombinant human AREG, BTC, and EREG were obtained from R&D Systems.

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

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: 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