Journal: Cell reports

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

doi: 10.1016/j.celrep.2013.01.028

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

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

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

Journal: Cell reports

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

doi: 10.1016/j.celrep.2013.01.028

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

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

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

Journal: Cell reports

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

doi: 10.1016/j.celrep.2013.01.028

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

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

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

Journal: Cell reports

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

doi: 10.1016/j.celrep.2013.01.028

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

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

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

Journal: Cell reports

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

doi: 10.1016/j.celrep.2013.01.028

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

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

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

Journal: BMC Ophthalmology

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

doi: 10.1186/s12886-022-02417-8

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

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

Techniques:

Journal: BMC Ophthalmology

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

doi: 10.1186/s12886-022-02417-8

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

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

Techniques: Significance Assay

Journal: BMC Ophthalmology

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

doi: 10.1186/s12886-022-02417-8

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

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

Techniques:

Journal: Bone

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

doi: 10.1016/j.bone.2017.10.012

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

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

Techniques: Expressing, Western Blot, Control, Gene Expression

Journal: Bone

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

doi: 10.1016/j.bone.2017.10.012

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

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

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

Journal: Middle East Fertility Society Journal

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

doi: 10.1186/s43043-025-00226-9

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

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

Techniques:

Journal: Middle East Fertility Society Journal

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

doi: 10.1186/s43043-025-00226-9

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

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

Techniques:

Journal: Biology of reproduction

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

doi: 10.1095/biolreprod.113.115311

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

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

Techniques: Glycoproteomics, Control, Staining, Fluorescence, Expressing

Journal: 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

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: International Journal of Molecular Sciences

Article Title: Rifaximin Attenuates Liver Fibrosis and Hepatocarcinogenesis in a Rat MASH Model by Suppressing the Gut–Liver Axis and Epiregulin–IL-8-Associated Angiogenesis

doi: 10.3390/ijms26146710

Figure Lengend Snippet: Effect of rifaximin on epiregulin and intrahepatic angiogenesis in CDAHFD-fed rats. ( A , B ) Hepatic expression of epiregulin at ( A ) protein and ( B ) mRNA levels. ( C ) Pearson’s correlation between Ereg and Lbp mRNA levels in all experimental rats. ( D ) Hepatic mRNA level of IL-8 . ( E ) Pearson’s correlation between IL-8 and Ereg mRNA levels in all experimental rats. ( F ) Representative microphotographs of CD34 staining of liver tissue. ( G ) Quantification of CD34-stained neovascularization in a high-power field. ( H ) Hepatic mRNA level of proangiogenic markers ( Pecam1 , Vcam1 , Flt1 , and Kdr ). Gapdh was used as an internal control for qRT-PCR ( B , D , H ). Data are the mean ± SD ( n = 10). Quantitative values are indicated as fold changes to the values of the CS+Veh group ( B , D , G , H ). * p < 0.05, ** p < 0.01, significant difference between groups determined by Student’s t -test.

Article Snippet: The epiregulin level in rat liver tissue was measured using a Rat Epiregulin ELISA Kit (Novus Biologicals, Centennial, CO, USA) as per the manufacturer’s protocol.

Techniques: Expressing, Staining, Control, Quantitative RT-PCR

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: Hepatobiliary Surgery and Nutrition

Article Title: EREG-secreting THBS1 + tissue monocytes are recruited by C5a to promote rapid liver regeneration in patients and mice during the ALPPS procedure

doi: 10.21037/hbsn-24-391

Figure Lengend Snippet: Proliferation phenotype of THBS1 + tissue monocytes. (A) t-SNE plot of the distribution of the Mo/Mφ population at ALPPS stage II. Results are color-coded according to the growth factor gene expression level. MP, Mo/Mφ population. (B) Violin and t-SNE plots of the EREG expression in THBS1 + tissue monocytes, i.e., THBS1 + tissue monocytes. (C) Network of hepatocyte receptor interactions (left panel) and bubble plot of monocyte cluster ligand and hepatocyte receptor interactions (right panel). (D) Bubble plot showing the expression of receptors in monocyte clusters. (E) Boxplot representing a comparison of the peak concentrations of EREG in the plasma of patients at ALPPS stages I and II. *, P<0.05. (F) Line chart showing the OD450 values of hepatocytes in the EREG group and control group following CCK-8 incubation. (G) In the liver tissue from the murine model of ALPPS, regeneration was impaired after systemic neutralization of EREG. (H) Survival analysis of EREG rescue versus vehicle treatment for mice with PHLF. *, P<0.05; **, P<0.01; ***, P<0.001 (actual P values were reported in results). ALPPS, associating liver partition and portal vein ligation for staged hepatectomy; CCK-8, Cell Counting Kit-8; EREG, epiregulin; FLR, future liver remnant; Mo, monocytes; Mφ, macrophages; MP, Mo/Mφ population; PHLF, post-hepatectomy liver failure; TM, tissue monocyte; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: The cells were resuspended in minimum essential medium (MEM) containing 10% FBS with recombinant EREG (5 μg/mL; rmEpiregulin/CF, 1068-EP-050/CF, R&D Systems), recombinant AREG (5 μg/mL; rmAmphiregulin/CF, 989-AR-100/CF, R&D Systems), recombinant EREG plus recombinant AREG (5 μg/mL), or vehicle control and inoculated into a 96 well plate with 1,000 cells per well.

Techniques: Gene Expression, Expressing, Comparison, Clinical Proteomics, Control, CCK-8 Assay, Incubation, Neutralization, Ligation, Cell Counting

Journal: Hepatobiliary Surgery and Nutrition

Article Title: EREG-secreting THBS1 + tissue monocytes are recruited by C5a to promote rapid liver regeneration in patients and mice during the ALPPS procedure

doi: 10.21037/hbsn-24-391

Figure Lengend Snippet: THBS1 + tissue monocytes were recruited through C5a. (A) Multiplexed immunofluorescence staining, showing the distribution of C5aR1 + monocytes (CD14 + C5aR1 + ). (B) Immunohistochemical staining of C5a at ALPPS stage I (upper left) and stage II (lower left). The H-score shows the up-regulation of C5a expression in hepatocytes from ALPPS stage I to stage II. *, P<0.05. (C) Liver samples from the murine ALPSS model on POD1 and POD5 (left). The boxplot represents comparisons of the FLR to body weight ratio between the control group and PMX-53 group on POD1 and POD5. (D) Immunohistochemical staining of Ki-67 for the control group and PMX-5 group on POD1 and POD5 (left). The boxplot shows comparisons of the ratios of Ki-67 + cells between the control and PMX-53 group on POD1 and POD5 (right). (E) Boxplot showing the EREG expression in the control and PMX-53 group on POD1 and POD5. (F) Schematic diagram of the ALPPS-induced liver regeneration. *, P<0.05 (actual P values were reported in results). ALPPS, associating liver partition and portal vein ligation for staged hepatectomy; EREG, epiregulin; IF, immunofluorescence; IHC, immunohistochemistry; FLR, future liver remnant; POD, post-operation day.

Article Snippet: The cells were resuspended in minimum essential medium (MEM) containing 10% FBS with recombinant EREG (5 μg/mL; rmEpiregulin/CF, 1068-EP-050/CF, R&D Systems), recombinant AREG (5 μg/mL; rmAmphiregulin/CF, 989-AR-100/CF, R&D Systems), recombinant EREG plus recombinant AREG (5 μg/mL), or vehicle control and inoculated into a 96 well plate with 1,000 cells per well.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Expressing, Control, Ligation, Immunohistochemistry