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: 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