Journal: Cell Death & Disease

Article Title: mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation

doi: 10.1038/s41419-022-05293-8

Figure Lengend Snippet: A Heatmap of the expression levels of ErbB signaling pathway signature genes in the vagina of control and Rptor cKO mice in the presence or absence of E2 administration. B qPCR was performed to verify the genes in the frame in A. n = 7 (OVX control mice), n = 5 (OVX Rptor cKO mice), n = 6 (OVX control mice treated with E2), n = 5 (OVX Rptor cKO mice treated with E2). Values are expressed as the mean ± SEM. C Representative images of the immunofluorescence staining of EREG in the vagina in OVX control and Rptor cKO mice administrated with or without E2. Nuclei were stained with DAPI. Microscopy with magnification ×20. Scale bars: 75 μm. D OVX control and Rptor cKO mice were administrated with E2 and/or EREG. The vaginas were harvested for PAS staining, Ki67 immunofluorescence staining, and TUNEL staining. In PAS staining, nuclei were stained with hematoxylin. Scale bars: 50 μm. In Ki67 immunofluorescence staining and TUNEL staining, nuclei were stained with DAPI. Microscopy with magnification ×20. Scale bars: 75 μm. E Expression levels of Krt6a , Krt6b , Krt10 , Krt13 , Krt16 in the vagina of the mice were quantified using qPCR. n = 6 (OVX control mice treated with E2), n = 5 (OVX Rptor cKO mice treated with E2), n = 4 (OVX Rptor cKO mice treated with E2 and EREG). Values are expressed as the mean ± SEM. F The vaginas as indicated in D were harvested for immunofluorescence staining of YAP1, nuclei were stained with DAPI. n = 6 (OVX control mice treated with E2), n = 5 (OVX Rptor cKO mice treated with E2), n = 4 (OVX Rptor cKO mice treated with E2 and EREG). The experiments were repeated three times. Microscopy with magnification ×20. Scale bars: 75 μm.

Article Snippet: For immunofluorescence staining, sections were incubated with the following primary antibodies: anti-Raptor (sc-81537, Santa Cruz Biotechnology), anti-Phospho-S6 Ribosomal Protein (Ser235/236) (62016, Cell Signaling Technology), anti-PR (9856 S, Cell Signaling Technology), anti-ER-alpha Ab (ab32063, Abcam), anti-Ki67 Ab (ab15580, Abcam), anti-YAP1 Ab (13584-1-AP, Proteintech), anti-EREG Ab (MAB1068, R&D Systems) overnight at 4 °C.

Techniques: Expressing, Control, Immunofluorescence, Staining, Microscopy, TUNEL Assay

Journal: Cell Death & Disease

Article Title: mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation

doi: 10.1038/s41419-022-05293-8

Figure Lengend Snippet: A mTORC1 signaling participates in the proliferation and differentiation of vaginal epithelium by promoting the expression level of PR and EREG-YAP1 in Rptor fl/fl mice. B Loss of Rptor compromises the estrogen-induced proliferation and differentiation of vaginal epitheliums through down-regulating the expression of PR and EREG.

Article Snippet: For immunofluorescence staining, sections were incubated with the following primary antibodies: anti-Raptor (sc-81537, Santa Cruz Biotechnology), anti-Phospho-S6 Ribosomal Protein (Ser235/236) (62016, Cell Signaling Technology), anti-PR (9856 S, Cell Signaling Technology), anti-ER-alpha Ab (ab32063, Abcam), anti-Ki67 Ab (ab15580, Abcam), anti-YAP1 Ab (13584-1-AP, Proteintech), anti-EREG Ab (MAB1068, R&D Systems) overnight at 4 °C.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Epiregulin expression and secretion is increased in castration-resistant prostate cancer

doi: 10.3389/fonc.2023.1107021

Figure Lengend Snippet: EREG expression and secretion is higher in CRPC in comparison to LNCaP cells (CSPC). (A) The expression of EREG after ADT treatment was assumed to be increased according to preliminary data. Analysis of EREG expression using qRT-PCR show an increased (p < 0.0001) EREG expression in CRPC cells in comparison to castration sensitive LNCaP cells. (B) Flow cytometry analysis shows the presence of EREG (dark) on the cell surface of CSPC and CRPC cell lines. Goat-IgG (light) is used as a control. Histograms show representative flow cytometry results for CSPC and CRPC cell lines using EREG antibody and goat-IgG. (C) The ratio is determined from the mean of the fluorescence intensity of the sample compared to the control. EREG protein on cell surface of 22Rv1 (p=0.0043) and LNCaP EnzR (p < 0.0001) cells is significantly increased in comparison to CSPC cells whereas EREG protein on DU145 (p=0.55) and PC3 (p=0.29) is slightly, but not significant increased compared to CSPC cells. (D) PCa cell culture supernatant was collected and EREG protein was investigated by sandwich ELISA. Whereas LNCaP and 22Rv1 cells secrete no EREG protein, it was detected in DU145 (330 pg/ml), PC3 (66 pg/ml) and LNCaP EnzR (37 pg/ml) supernatant. All graphs show the mean and SD from four independently performed experiments (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: The protein content of EREG in the cell culture supernatant was determined by sandwich ELISA using the Human Epiregulin DuoSet ELISA (R&D Systems, Minneapolis, Minnesota, US) according to the manufacturer’s instructions.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Flow Cytometry, Control, Fluorescence, Cell Culture, Sandwich ELISA

Journal: Frontiers in Oncology

Article Title: Epiregulin expression and secretion is increased in castration-resistant prostate cancer

doi: 10.3389/fonc.2023.1107021

Figure Lengend Snippet: Amount of EREG correlates with increasing aggressiveness of PCa samples. TMA with 196 samples were immunohistochemically stained with EREG antibody. (A) Representative overview and detail pictures of EREG staining in PCa samples for different categories of IRS (Overview pictures: Magnification 200x, scale bar 200 µm; Detail pictures in black boxes: Magnification 630x, scale bar 100 µm). Samples with an IRS of 0 exhibit no expression of EREG, while samples with an IRS of 1 show a weak EREG expression. An IRS of 2 indicates an intermediate EREG expression and an IRS of 3 a strong expression. (B) The pie chart shows the distribution of cases split by IRS for EREG. (C) Violin plot with correlation of EREG IRS and preoperative PSA levels (median: dashed line, 95% quartile: dotted line). The EREG IRS tends to correlate positively with PSA level without significance (p=0.29; ns, not significant). (D–H) Bubble plots show the distribution of EREG IRS in relation to (D) PCa related death, (E) PCa grading, (F) tumor recurrence, (G) CHGA IRS and (H) SYP IRS. The size of the bubbles indicates the relative number of cases, while the numbers to the right of the bubbles represent the absolute number of cases. CHGA and SYP IRS were determined in the same manner as for EREG.

Article Snippet: The protein content of EREG in the cell culture supernatant was determined by sandwich ELISA using the Human Epiregulin DuoSet ELISA (R&D Systems, Minneapolis, Minnesota, US) according to the manufacturer’s instructions.

Techniques: Staining, Expressing

Journal: Frontiers in Oncology

Article Title: Epiregulin expression and secretion is increased in castration-resistant prostate cancer

doi: 10.3389/fonc.2023.1107021

Figure Lengend Snippet: Elevated phosphorylation of SMAD2/3 seems to enhance EREG expression in PCa cells. (A) Column graph shows ERK1/2 phosphorylation at Thr202/Tyr204 in PCa cell lines LNCaP, 22Rv1, DU145 and PC3. ERK1/2 phosphorylation in 22Rv1 and DU145 cells are increased in comparison to LNCaP cells. Additionally, a representative immunoblot image for ERK1/2 phosphorylation in PCa cell lines is illustrated. (B) ERK1/2 phosphorylation at Thr202/Tyr204 of LNCaP in comparison to LNCaP EnzR cells and a representative immunoblot image are shown. Phosphorylation of ERK1/2 is enhanced in LNCaP EnzR cells. (C) The column graph shows the phosphorylation of transcription factor ETS1 at Thr38 in the different PCa cell lines without differences in ETS1 phosphorylation and expression. The adjacent representative immunoblot illustrates the result. (D) As revealed in the graph and the representative immunoblot, LNCaP EnzR exhibit no alteration in ETS1 phosphorylation at Thr38 compared with the parental LNCaP cells as well. (E) The bar graph and representative immunoblots shows the phosphorylation of transcription factor SMAD2/3 at Ser465, Ser467, Ser423, Ser425 in PCa cell lines. Compared to LNCaP cells, SMAD2/3 phosphorylation in 22Rv1 and DU145 is slightly increased, but PC3 cells show a noticeably higher phosphorylation of SMAD2/3. (F) SMAD2/3 phosphorylation of LNCaP EnzR cells is illustrated in a column graph and a representative immunoblot image. Phosphorylation in SMAD2/3 at Ser465, Ser467, Ser423, Ser425 is increased in LNCaP EnzR in comparison to parental LNCaP cells. All graphs show the mean and SD from four independently performed experiments. (G) Total EREG protein in cell culture supernatant of DU145 cells after TGF-β1 (p=0.2649) or TGF-β2 (p=0.0283) treatment is significantly elevated in comparison to untreated DU145 cells. All graphs show the mean and SD from four independent experiments (ns, not significant; *p < 0.05).

Article Snippet: The protein content of EREG in the cell culture supernatant was determined by sandwich ELISA using the Human Epiregulin DuoSet ELISA (R&D Systems, Minneapolis, Minnesota, US) according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Expressing, Comparison, Western Blot, Cell Culture

Journal: Frontiers in Oncology

Article Title: Epiregulin expression and secretion is increased in castration-resistant prostate cancer

doi: 10.3389/fonc.2023.1107021

Figure Lengend Snippet: EREG protein biosynthesis is post-transcriptionally regulated by miRNAs. (A) The expression of miR-19a and -19b that were assumed to be reduced according to preliminary data was assessed by qRT-PCR. MiR-19a (p=0.0001) and -19b (p=0.0053) were significantly reduced in LNCaP EnzR compared to parental LNCaP cells. (B) Predicted miRNA binding sites in the 3’UTR of EREG mRNA for miR-19a, -19b, -20b and the mutated miRNA binding sites are shown. The bold nucleotides in the sequences represent the miRNAs seed sequence and its corresponding binding sites. (C–E) For luciferase reporter assay, miRNA expression plasmids (control grey bar, miRNA black bar) were cotransfected with empty reporter plasmid (control), reporter gene construct containing wildtype EREG 3’UTR (WT) or reporter gene construct containing mutated EREG 3’UTR (MUT). The luciferase activity of the reporter gene plasmid coexpressed with control expression plasmid was set to 1. MiR-19a (p=0.0001), -19b (p=0.0006) and -20b (p<0.0001) significantly reduce luciferase activity of the reporter gene construct containing EREG 3’UTR in comparison to the control. After mutation of the seed sequence, the corresponding miRNAs are not able to reduce the luciferase activity. (F) Flow cytometry analysis using EREG antibody show the EREG presence on the cell surface of LNCaP EnzR cells transfected with miRNA expression plasmid. The histograms show representative flow cytometry analyses of LNCaP EnzR cells transfected with miRNA expression plasmid using EREG antibody (dark) and goat-IgG (light). (G) The ratio is determined from the mean of the fluorescence intensity of the specific EREG antibody compared to the control (goat-IgG). EREG protein on cell surface of LNCaP EnzR cells transfected with expression plasmids for miR-19a (p=0.0008), miR-19b (p=0.001) or miR-20b (p=0.0015) is significantly decreased in comparison to LNCaP EnzR cells transfected with control expression plasmid. All graphs show the mean and SD from four independent experiments (ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (H) Total EREG protein in cell culture supernatant of DU145 cells transfected with expression plasmids for miR-19a (p=0.0015), miR-19b (p=0.0029) or miR-20b (p=0.038) is significantly decreased in comparison to DU145 cells transfected with control expression plasmid. All graphs show the mean and SD from four independent experiments (ns, not significant; *p < 0.05; **p < 0.01).

Article Snippet: The protein content of EREG in the cell culture supernatant was determined by sandwich ELISA using the Human Epiregulin DuoSet ELISA (R&D Systems, Minneapolis, Minnesota, US) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Sequencing, Luciferase, Reporter Assay, Control, Plasmid Preparation, Construct, Activity Assay, Comparison, Mutagenesis, Flow Cytometry, Transfection, Fluorescence, Cell Culture

Journal: Frontiers in Oncology

Article Title: Epiregulin expression and secretion is increased in castration-resistant prostate cancer

doi: 10.3389/fonc.2023.1107021

Figure Lengend Snippet: Proteases, shedding the ectodomain of EREG are increased in CRPC compared to CSPC cells. (A) The dot plot depicts the ADAM17 expression of CSPC and CRPC cell lines using qRT-PCR analysis. 22Rv1, DU145, PC3 and LNCaP EnzR (p<0.0001) significantly express more ADAM17 compared to LNCaP cells. (B) Flow cytometry analysis using ADAM17 antibody show the ADAM17 presence on the cell surface of CSPC and CRPC cell lines. ADAM17 protein level on cell surface of PC3 (p<0.0001) and LNCaP EnzR (p < 0.0001) cells is increased in comparison to LNCaP cells whereas ADAM17 protein content on 22Rv1 and DU145 is not altered. (C) The pictures show representative flow cytometry analysis of CSPC and CRPC cell lines using ADAM17 antibody (dark) and mouse-IgG (light) as a control. MMP2 (D) and MMP9 (E) expression in CSPC and CRPC cell lines was determined by qRT-PCR. In comparison to LNCaP cells 22Rv1 (p=0.0057), PC3 (p=0.0002) and LNCaP EnzR (p < 0.0001) cells show an increased expression of MMP2. The MMP9 expression of 22Rv1 (p < 0.0001), DU145 (p < 0.0001), PC3 (p < 0.0001) and LNCaP EnzR (p < 0.0001) is enhanced compared to LNCaP cells. (F) Solid phase ELISA using a specific MMP2 antibody demonstrates the secretion of MMP2 in cell culture supernatant of PCa cell lines. The ELISA results reveal that MMP2 is secreted by all five PCa cell lines, but 22Rv1 (p=0.0021), DU145 (p=0.0026), PC3 (p < 0.0001) and LNCaP EnzR (p < 0.0001) cells secrete a higher amount of MMP2 protein in comparison to LNCaP cells. (G) PCa cell culture supernatant was collected and MMP9 protein was investigated by solid phase ELISA. All PCa cells secrete MMP9 protein, but the CRPC cell lines 22Rv1 (p < 0.0001), DU145 (p=0.0005), PC3 (p < 0.0001) and LNCaP EnzR (p < 0.0001) secrete more MMP9 protein than CSPC cell line LNCaP. All graphs show the mean and SD from four independent experiments (ns, not significant; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: The protein content of EREG in the cell culture supernatant was determined by sandwich ELISA using the Human Epiregulin DuoSet ELISA (R&D Systems, Minneapolis, Minnesota, US) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Comparison, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

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: 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: Cancer science

Article Title: Activation of epidermal growth factor receptor signaling by the prostaglandin E(2) receptor EP4 pathway during gastric tumorigenesis.

doi: 10.1111/j.1349-7006.2011.01847.x

Figure Lengend Snippet: Fig. 3. (a) Representative photographs of the primary cultured gastric epithelial cells in matrigel from wild type (left) and K19-Wnt1 mice (right). Arrowheads indicate cystic structures, while arrows indicate clusters of dead cells. Bars indicate 500 lm. (b) Histology (top, H&E) and immunostaining with anti-active b-catenin antibody (bottom) of cystic structures. Inset indicates nuclear accumulation of active b-catenin in the epithelial cells. (c) Relative expression of epidermal growth factor receptor (EGFR) ligands and a disintegrin and metalloproteinases (ADAMs) in gastric epithelial cells cultured in matrigel with the indicated treatment (mean ± SD). Asterisk indicates P < 0.05 versus the level in control cells cultured in EGF ()) medium. (d) The mean number of cystic structures >75 lm in diameter in matrigel of the amphiregulin-treated (Areg), epiregulin-treated (Ereg) and control (cont) gastric epithelial cells (mean ± SD). Asterisks indicate P < 0.05.

Article Snippet: The primary cultured cells were stimulated with mouse recombinant amphiregulin and epiregulin (R&D) at 20 and 1 ng ⁄ mL, respectively, and the mean number of cystic structures >75 lm in diameter per microscopic field was calculated at day 5.

Techniques: Cell Culture, Immunostaining, Expressing, Control

Journal: International journal of oncology

Article Title: Use of multiple biomarkers for the localization and characterization of colon cancer stem cells by indirect immunocytochemistry.

doi: 10.3892/ijo.2012.1430

Figure Lengend Snippet: Figure 4. Staining of tumor sections with either antibodies to KRAS or to epiregulin. (A) Cluster of stem cells of poorly differentiated tumor (stage II) stained with antibodies to KRAS. The staining is essentially in the cytoplasm and the nuclei of the stem cells (double arrows). Some of the nuclei are enlarged (single arrows). There is also occasionally weak labeling in the cytoplasm of other cells. Original magnification, x400. (B) Stained section through the villi of the normal colon with antibodies to epiregulin. Staining of the cytoplasm of the villi cells is evident (double arrows), staining of a distinct population of the cells in between the villi is also evident (single arrows). Original magnification, x400. (C) Highly differentiated carcinoma cells stained with antibodies to epiregulin. The cytoplasm of the cells is weakly stained (double arrows). Original magnification, x400. (D) Moderately differentiated colon cancer cells. The cytoplasm of the cells is stained more intensively than the cytoplasm of the highly differentiated colon tumors (double arrows). Original magnification, x400.

Article Snippet: The affinity purified antibody, anti-human epiregulin, was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Labeling

Journal: International journal of oncology

Article Title: Use of multiple biomarkers for the localization and characterization of colon cancer stem cells by indirect immunocytochemistry.

doi: 10.3892/ijo.2012.1430

Figure Lengend Snippet: Figure 5. Staining of 2 colon cancer sections with antibodies to epiregulin. (A) Poorly differentiated tumor cells stained with antibodies to epiregulin. Cluster of stem cells is intensively stained (double arrows). Large nuclei within the clustered cells are evident (single arrows). Some cells of the tumor (Tu) are also weakly labeled in the cytoplasm. Original magnification, x400. (B) Invasive tumor (stage IV) in between fat cells (FC) distinct tumor cells are clearly labeled with epiregulin antibodies (arrows). Original magnification, x400.

Article Snippet: The affinity purified antibody, anti-human epiregulin, was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Labeling

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