Journal: Nature neuroscience

Article Title: A glycolytic shift in Schwann cells supports injured axons

doi: 10.1038/s41593-020-0689-4

Figure Lengend Snippet: a, Top: Scheme of metabolomic analysis using extracts from nerve segments. Bottom: Concentrations of key energy metabolism intermediates in control and axotomized nerve segments from C57Bl/6J mice (F6P/G6P: fructose-6-phosphate/glucose-6-phosphate. FBP: fructose-1,6-bisphosphate. GI-OH3P: Glyceraldehyde-3-phosphate. 2PG/3PG: 2-phosphoglycerate/3-phosphoglycerate. LACT: lactate) (Error bars represent s.e.m. n=5 mice per condition and metabolite). b, Top: Scheme of extracellular flux analysis of SCs purified from C57Bl/6J mouse nerves. Bottom: Box and whiskers plot (maximum, 25th percentile, median, 75th percentile, minimum) shows glycolytic activity parameters as assessed by extracellular acidification rate (ECAR) measurements in control mouse SCs and cells with Nrg1-induced ErbB2 activation (n=9 well preparations per condition, *P=0.0080, **P=0.0464, ***P<0.0001). c, Western blot analysis (cropped blot images) of control- and Nrg1-treated C57Bl/6J mouse SCs probed with the indicated antibodies. (n=6 independent pair preparations (2 separate dishes) for PFKFB3, and n=3 independent pair preparations (2 separate dishes) for LDHA quantification). d, Intracellular and extracellular (supernatant) lactate concentrations from control and Nrg1-treated mouse SC preparations normalized to cell number and cellular protein. Note decreased intracellular and increased extracellular lactate levels in SCs treated with Nrg1 for 24h, indicating greatly increased lactate extrusion (Error bars represent s.e.m. n=3 well preparations from 3 independent experiments per condition).

Article Snippet: SCs were subsequently control-treated or treated with 200 ng/ml recombinant Nrg1 (R&D Systems, 396-HB-050) for 24h, collected in RIPA buffer containing phosphatase and protease inhibitors, and then processed for protein analysis and western blotting using standard procedures.

Techniques: Control, Purification, Activity Assay, Activation Assay, Western Blot

Journal: Oncology Reports

Article Title: Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells

doi: 10.3892/or.2015.4126

Figure Lengend Snippet: Effect of TGF-β and HB-EGF gene silencing on HCC cell lines co-cultured with HSCs. HSCs were treated with TGF-β1/2/3 and HB-EGF siRNA for 5 h. Then, HCC cell lines were co-cultured with siRNA-treated HSCs for 48 h. (A) Cell proliferation activity was evaluated by MTT assay. Data represent the mean ± SD of >3 independent experiments, each performed in triplicate. n.s., p>0.05; * p<0.05 (vs. data for control siRNA treated group); Student's t-test. (B) Cell migration activity was assessed by an in vitro wound healing assay. Data represent the mean ± SD of >3 independent experiments, each performed in triplicate. n.s., p>0.05; * p<0.05 (vs. data for control siRNA treated group); Student's t-test. (C) HCC cells were isolated and lysed. Immunoblot analysis was performed using antibodies against NICD and Hes1. Immunoblotting for β-actin was used to verify equal loading of cellular proteins. Representative blots of >3 independent experiments are shown. HCC, hepatocellular carcinoma; HSCs, hepatic stellate cells.

Article Snippet: TWNT-1 was transfected with scrambled negative control siRNA and TGF-β1/2/3, and HB-EGF siRNA, respectively (sc-37007, sc-44146 and sc-39420; SCBT). siRNAs were transfected into cells using a siRNA transfection reagent and siRNA transfection medium (sc-45064; SCBT).

Techniques: Cell Culture, Activity Assay, MTT Assay, Control, Migration, In Vitro, Wound Healing Assay, Isolation, Western Blot

Journal: Oncology Reports

Article Title: Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells

doi: 10.3892/or.2015.4126

Figure Lengend Snippet: Effect on EMT and stemness of TGF-β and HB-EGF gene silencing in HCC cell lines co-cultured with HSCs. (A) HSCs were treated with TGF-β1/2/3 and HB-EGF siRNA for 5 h. Then, HCC cell lines were co-cultured with siRNA-treated HSCs for 72 h. HCC cells were collected and washed in PBS without Ca 2+ /Mg 2+ and incubated with APC-conjugated anti-EpCAM antibody. The cells were analyzed with MACSQuant flow cytometer. Representative plots from >3 independent experiments are shown. (B) HSCs were treated with TGF-β1/2/3 and HB-EGF siRNA for 5 h. Then, HCC cell lines were co-cultured with siRNA-treated HSCs for 72 h. HCC cells were collected and washed in PBS without Ca 2+ /Mg 2+ and incubated with APC-vio770-conjugated anti-E-cadherin and PE-conjugated anti-N-cadherin. The cells were analyzed with the flow cytometer. Representative plots from >3 independent experiments are shown. EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; HSCs, hepatic stellate cells; EpCAM, epithelial cell adhesion molecule.

Article Snippet: TWNT-1 was transfected with scrambled negative control siRNA and TGF-β1/2/3, and HB-EGF siRNA, respectively (sc-37007, sc-44146 and sc-39420; SCBT). siRNAs were transfected into cells using a siRNA transfection reagent and siRNA transfection medium (sc-45064; SCBT).

Techniques: Cell Culture, Incubation, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 2. Induction of HBEGF ectoshedding by mechanical strain and ET-1. Cardiac myocytes were subjected to a static or strain environment for 0–30 min (panel A) or ET-1 (107 M) for 5 min (panel B), after which medium was collected and concentrated. HBEGF was detected by immunoprecipitation and immunoblotting. Data are ex- pressed as means S.E., n 3. *, p 0.05; **, p 0.01 versus static control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 3. EGFR ligands activate the BNP promoter. Cardiac myo- cytes were co-transfected with 0.5 g of 1595 hBNP-luciferase and 0.05 g of actin -galactosidase. 18–24 h post-transfection, cells were deprived of serum for 24 h and then treated with 0–10 ng/ml EGF (panel A, n 4) or HBEGF (panel B, n 4) for 24 h. Cells were processed and assayed for luciferase activity. Data are expressed as means S.E. *, p 0.05 versus control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 5. Induction of cardiac myo- cyte hypertrophy by HBEGF. After 24 h of culture, cells were treated with HBEGF (1 or 10 ng/ml) or ET-1 (107 M) for 48 h. Panel A, HBEGF stimulates pro- tein synthesis in cultured neonatal rat ventricular myocytes. Following treat- ment, cells were pulsed with [3H]leucine for 4 h, and incorporation of radioactivity into acid-insoluble protein was measured (n 4). Panels B and C, HBEGF in- creases surface area (panel B) and sarco- meric organization (panel C) of cultured neonatal rat ventricular myocytes. Fol- lowing treatment, cells were fixed with 3.7% paraformaldehyde, stained with monoclonal antibody against rat sarcom- eric -actinin, and viewed using fluores- cence microscopy. Panel B, cell surface areas of individual cells were quantified as described under “Experimental Proce- dures” (n 20). Data are expressed as the means S.E. *, p 0.05; **, p 0.01 versus control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Radioactivity, Staining, Microscopy, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 6. Role of endothelin-1 in stretch-dependent BNP promoter ac- tivation and HBEGF ectoshedding. Panels A and C, cardiac myocytes were co-transfected with 0.5 g of 1595 hu- man BNP-luciferase and 0.05 g of actin -galactosidase. 18–24 h post-transfec- tion, cells were deprived of serum for 24 h and then pretreated with the ETA-recep- tor antagonist BQ-610 (1 M; panel A) or the EGFR inhibitor AG-1478 (250 nM; panel C) for 1 h. Myocytes were then sub- jected to a static or strain environment (panel A) or treated with endothelin-1 (107 M; panel C) for 48 h, processed, and assayed for luciferase activity (n 3). Panel B, cells were pretreated with BQ- 610 (1 M) for 1 h and then subjected to a static or stretch environment for 0–5 min. Medium was collected and concentrated, and HBEGF was detected by immunopre- cipitation and immunoblotting (n 3). Data are expressed as means S.E. **, p 0.01; ***, p 0.001 versus control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Activity Assay, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 7. Metalloproteinase activity is required for strain-de- pendent effects. Panel A, cardiac myocytes were pretreated with a metalloproteinase inhibitor, GM-6001 (4 M), for 1 h and then subjected to a static or strain environment for 0–5 min. Medium was collected and concentrated, and HBEGF was detected by immunoprecipitation and immunoblotting (n 3). Panel B, cardiac myocytes were co-transfected with 0.5 g of 1595 hBNP-luciferase and 0.05 g of actin -galacto- sidase. 18–24 h post-transfection, cells were deprived of serum for 24 h and pretreated with the GM-6001 (4 M) or vehicle for 1 h. Myocytes were then subjected to a static or strain environment for 48 h, pro- cessed, and assayed for luciferase activity (n 3). Data are expressed as means S.E. **, p 0.01 versus static control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Transfection, Luciferase, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 8. Role of ROS in strain- dependent effects. Panel A, antioxidant blocks strain-dependent HBEGF ectoshedding. Cardiac myocytes were pre- treated with an antioxidant, N-acetylcys- teine (NAc; 10 mM), for 1 h and then sub- jected to a static or strain environment for 0–5 min. Medium was collected and con- centrated, and HBEGF was detected by immunoprecipitation and immunoblot- ting. Panels B–D, effect of antioxidant on strain-dependent (panel B) and ET-1-de- pendent (panel C) BNP promoter activa- tion or on strain-dependent ET-1 pro- moter activation (panel D). Cardiac myocytes were co-transfected with 0.5 g of 1595 hBNP-luciferase or 1315 rat ET-1-luciferase and 0.05 g of actin -ga- lactosidase. 18–24 h post-transfection, cells were deprived of serum for 24 h and then pretreated NAc (10 mM) for 1 h. Myo- cytes were subjected to a static or strain environment (panels B and D) or endothe- lin-1 (107 M; panel C) for 48 h, processed, and assayed for luciferase activity. Data are expressed as means S.E. (n 3). NS, not significant; *, p 0.05; **, p 0.01 versus control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Western Blot, Activation Assay, Transfection, Luciferase, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 9. Role of NAD(P)H oxidase in strain-dependent effects. Panel A, Effect of NAD(P)H oxidase inhibition on strain-dependent HBEGF ectoshedding. Cardiac myocytes were pretreated with the NAD(P)H inhibitor and apocynin (100 M) for 1 h and then subjected to a static or strain environment for 0–5 min. Medium was collected and concentrated. HBEGF was detected by immunoprecipitation and immunoblotting. Panels B–D, role of NAD(P)H oxidase in strain-dependent (panel B) and ET-1-dependent (panel C) BNP promoter activation or on strain-dependent ET-1 promoter activation (panel D). Cardiac myocytes were co-transfected with 0.5 g of 1595 hBNP-luciferase or 1315 rat ET-1-luciferase and 0.05 g of actin -galactosidase. 18–24 h post-transfection, cells were deprived of serum for 24 h and then pretreated with apocynin (100 M) for 1 h. Myocytes were subjected to a static or strain environment (panels B and D) or endothelin-1 (107 M; panel C) for 48 h, processed, and assayed for luciferase activity. Data are expressed as means S.E. (n 3). NS, not significant; *, p 0.05; **, p 0.01 versus control.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Inhibition, Immunoprecipitation, Western Blot, Activation Assay, Transfection, Luciferase, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Role of the Epidermal Growth Factor Receptor in Signaling Strain-dependent Activation of the Brain Natriuretic Peptide Gene

doi: 10.1074/jbc.m309227200

Figure Lengend Snippet: FIG. 10. Schematic representation of signaling cascade linking mechanical strain to activation of the BNP gene promoter. Strain activates NAD(P)H oxidase, which generates ROS. This pro- motes production of ET-1, which stimulates metalloproteinase-medi- ated cleavage of pro-HBEGF to HBEGF, activates the EGFR and sub- sequently leads to an increase in hBNP promoter activity.

Article Snippet: Materials—Recombinant human HBEGF was from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Activity Assay

Journal: iScience

Article Title: 2′-Fucosyllactose transactivates EGF receptor in intestinal epithelial cells for prevention of colitis in adulthood

doi: 10.1016/j.isci.2025.114308

Figure Lengend Snippet: 2′-FL transactivates EGFR in IECs via stimulating the release of HB-EGF from the cell membrane to bind to EGFR (A and C) Cells were treated with 2′-FL at indicated concentrations or 20 μg/mL of lactose or glucose for 2 h (A), or 2′-FL at 20 μg/mL for 2 h with and without 1-h pretreatment of HB-EGF neutralizing antibody at 500 ng/mL that was present with 2′-FL treatment (C). Total cellular lysates were prepared for Western blot analysis of the phosphorylated and total levels of EGFR and ERK1/2. β-actin blot was used as the protein loading control. (B) Cells were treated with 2′-FL at 20 μg/mL for indicated times. Cell culture supernatants were collected for ELISA analysis of HB-EGF levels. Data are presented as pg of HB-EGF in culture supernatant from 105 cells. (D) YAMC cells were treated with 2′-FL, lactose, or glucose at 20 μg/mL for 2 h. At the end of the treatment, the fluorogenic peptide substrate (4 μM) was added to cell culture medium and incubated for 2 h. The fluorescent intensity in supernatants was measured at 30, 60, 90, and 120 min after reaction. Data are presented as relative fluorescence unit (RFU)/minute in the indicated reaction period from 104 cells. Images in (A) and (C) represent data from at least three independent experiments. In (B) and (D), data were triplicated in each experiment, and the symbol represents the average data from individual experiment. Statistical significance was determined using unpaired t -tests and one-way ANOVA analysis of variance. ∗ p < 0.05 compared to the control group of the same cell line in (B). ∗ p < 0.05 compared to the control, glucose, and lactose groups in the same reaction period in (D). Data are represented as mean ± SD.

Article Snippet: Supernatants from YAMC and EGFR −/− MCE cultures were collected to examine levels of HB-EGF and TGFα using mouse HB-EGF DuoSet ELISA kit (#DY8239–05, R&D Systems, Inc.) and mouse TGFα ELISA kit (#MBS2701286, MyBoSource Inc), respectively, according to the manufacturer’s instructions.

Techniques: Membrane, Western Blot, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Fluorescence