Journal: eLife

Article Title: Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation

doi: 10.7554/eLife.50990

Figure Lengend Snippet: Neutrophils were incubated with media or a concentration range of gefitinib ( A ), lapatinib ( A ), sapatinib ( A ), CP-724714 ( B ), erbstatin (Erb, C ) or tyrphostin AG825 (Tyr, D ) for 6 hr. Stars represent significant difference compared to DMSO control (indicated by ‘0’ in B-D). Neutrophils from COPD patients (open bars) and age-matched healthy control subjects (black bars) were incubated with DMSO or a concentration range of erbstatin ( E ) or tyrphostin AG825 ( F ) for 6 hr. Apoptosis was assessed by light microscopy. The data are expressed as mean percentage apoptosis ± SEM from 3 ( B, D ), 4 ( A,C ), 10 (E,F COPD), or 7 (E,F HC) independent experiments using different neutrophil donors. Statistical significances between control and inhibitor was calculated by one-way ANOVA with Dunnett’s post-test, indicated as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: C57BL/6 mice (female, 9–10 weeks old) were nebulised with LPS (3 mg per group of 8 mice) ( Pseudomonas aeruginosa , Sigma-Aldrich) and immediately injected intraperitoneally (i.p.) with either Tyrphostin AG825 (Tocris Bioscience) at 20 mg/Kg in 10% DMSO v/v in vegetable oil (eight mice, treatment group) or an equivalent volume of 10% DMSO v/v in vegetable oil (eight mice, control group) ( ; ).

Techniques: Incubation, Concentration Assay, Control, Light Microscopy

Journal: eLife

Article Title: Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation

doi: 10.7554/eLife.50990

Figure Lengend Snippet: Neutrophils were incubated with DMSO or 20 µM or 40 µM erbstatin (Erb, A and B ) or tyrphostin AG825 (Tyr, C ) for 6 hr. Apoptosis was assessed by Annexin V/TO-PRO-3 staining by flow cytometry and the percentage apoptosis calculated as Annexin V single plus Annexin V/TO-PRO-3 dual positive events. ( A ) Representative quadrant plot of Erbstatin-treated neutrophils showing distribution of Annexin V and TO-PRO-3 positive events. ( D–E ) Neutrophils were incubated with DMSO or Erbstatin [40 µM] in the presence or absence of the pan caspase inhibitor, Q-VD-OPh [1 µM] for 6 hr ( D ) or 20 hr ( E ). Apoptosis was assessed by light microscopy. The data are expressed as mean percentage apoptosis ± SEM from 3 ( B ), 4 (C and E), or 5 ( D ) independent experiments. Statistical differences were calculated by one-way ANOVA (with Dunnett’s post test ( B–C ) and two-way ANOVA with Sidak ( D–E ) post-tests) and indicated as *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Article Snippet: C57BL/6 mice (female, 9–10 weeks old) were nebulised with LPS (3 mg per group of 8 mice) ( Pseudomonas aeruginosa , Sigma-Aldrich) and immediately injected intraperitoneally (i.p.) with either Tyrphostin AG825 (Tocris Bioscience) at 20 mg/Kg in 10% DMSO v/v in vegetable oil (eight mice, treatment group) or an equivalent volume of 10% DMSO v/v in vegetable oil (eight mice, control group) ( ; ).

Techniques: Incubation, Staining, Flow Cytometry, Light Microscopy

Journal: eLife

Article Title: Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation

doi: 10.7554/eLife.50990

Figure Lengend Snippet: Neutrophils were incubated with DMSO, Erbstatin [Erb, 40 µM] ( A ) or tyrphostin AG825 [Tyr, 50 µM] ( B ) in the presence of DMSO or N 6 -MB-cAMP [500 µM and 1 mM] for 20 hr. Neutrophils isolated from COPD patients were incubated with DMSO or tyrphostin AG825 [50 µM] in the presence of DMSO or N 6 -MB-cAMP [500 µM] for 20 hr ( C ). Neutrophils isolated from COPD patients and age-matched healthy control subjects (HC) were incubated with DMSO, erbstatin ( D ) [20, 40 µM] or tyrphostin AG825 ( E ) [25, 50 µM] in the presence or absence of GMCSF [50 u/mL] for 20 hr. Apoptosis was assessed by light microscopy. The data are expressed as mean percentage apoptosis ± SEM from 4 to 6 independent experiments. ( F ) Neutrophils were incubated with DMSO or tyrphostin AG825 [Tyr, 50 µM] for 60 min before the addition of GMCSF [50 u/mL] for 15 or 30 mins. ( G ) Neutrophils were incubated with DMSO, tyrphostin AG825 [50 µM] for 60 min before the addition of GMCSF [50 u/mL] for a further 7 hr. Cells were lysed, subjected to SDS-PAGE electrophoresis and membranes probed for p-AKT, Mcl-1 or loading controls, AKT and P38. Images are representative of 3 independent experiments. Charts show densitometric values of 3 individual immunoblots and are expressed as a ratio of target (p-AKT or Mcl-1) over loading control (AKT or P38, respectively). Statistically significant differences were calculated by one-way ANOVA with Sidak post-test ( A–C, F–G ) or two-way ANOVA with Sidak post-test ( D–E ) and indicated as *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: C57BL/6 mice (female, 9–10 weeks old) were nebulised with LPS (3 mg per group of 8 mice) ( Pseudomonas aeruginosa , Sigma-Aldrich) and immediately injected intraperitoneally (i.p.) with either Tyrphostin AG825 (Tocris Bioscience) at 20 mg/Kg in 10% DMSO v/v in vegetable oil (eight mice, treatment group) or an equivalent volume of 10% DMSO v/v in vegetable oil (eight mice, control group) ( ; ).

Techniques: Incubation, Isolation, Control, Light Microscopy, SDS Page, Electrophoresis, Western Blot

Journal: eLife

Article Title: Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation

doi: 10.7554/eLife.50990

Figure Lengend Snippet: C57BL/6 mice were nebulized with LPS and immediately injected intraperitoneally with either 10% DMSO (control, n = 8) or 20 mg/Kg tyrphostin AG825 (Tyr, n = 8). After 48 hr the mice were sacrificed and subjected to bronchoalveolar lavage. Percentage neutrophils (A, closed icons) and macrophages (A, open icons) and absolute numbers of neutrophils (B, closed icons) and macrophages (B, open icons) in BAL were calculated by haemocytometer and light microscopy. ( C ) Percentage neutrophil apoptosis (circles) and percentage neutrophil apoptosis calculated by also including numbers of apoptotic inclusions visualised within macrophages (triangles) was assessed by light microscopy. ( D ) Macrophages containing one or more apoptotic inclusions expressed as a percentage of all macrophages. Light microscopy image showing apoptotic inclusions within macrophages as indicated by black arrows ( E ). C57BL/6 mice were injected i.p. with 1 mg zymosan and 4 hr later injected i.p. with 20 mg/Kg tyrphostin AG825 (Tyr, n = 5) or 10% DMSO (Control, n = 5). At 20 hr mice were sacrificed and subjected to peritoneal lavage. ( F ) WBC, neutrophils and macrophages in blood were measured by a Sysmex cell counter. Total cells in peritoneal lavage were counted by flow cytometry ( G ) and KC, IL-6 ( H ) and IgM ( I ) measured in lavage by ELISA. At least two independent experimental replicates each processing 1–3 mice/group were performed. Statistical significance was calculated by Mann–Whitney U test ( A–D and G–I ) or one-way ANOVA with Sidak post-test ( F ) and indicated as *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: C57BL/6 mice (female, 9–10 weeks old) were nebulised with LPS (3 mg per group of 8 mice) ( Pseudomonas aeruginosa , Sigma-Aldrich) and immediately injected intraperitoneally (i.p.) with either Tyrphostin AG825 (Tocris Bioscience) at 20 mg/Kg in 10% DMSO v/v in vegetable oil (eight mice, treatment group) or an equivalent volume of 10% DMSO v/v in vegetable oil (eight mice, control group) ( ; ).

Techniques: Injection, Control, Light Microscopy, Flow Cytometry, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: eLife

Article Title: Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation

doi: 10.7554/eLife.50990

Figure Lengend Snippet: Tail fin transection was performed as indicated by the red line ( A , upper image). Zebrafish larvae ( mpx :GFP) were pre-treated at two dpf with DMSO, tyrphostin AG825 [Tyr, 10 µM] ( B , minimum n = 28 larvae per condition), or CP-724714 [10 µM] ( C , minimum n = 42 larvae per condition) for 16 hr followed by injury. egfra and erbb2 crispants were generated and injured at two dpf ( D , minimum n = 36 larvae per condition). The number of neutrophils at the site of injury was determined at 4 and 8 hpi by counting GFP-positive neutrophils. To enumerate neutrophils across the whole body, uninjured inhibitor treated larvae (three dpf) ( E , minimum n = 23 larvae per condition) or crispants (two dpf) ( F , minimum n = 28 larvae per condition) were imaged by fluorescent microscopy (A, lower image). Apoptosis was measured at the site of injury after 8 hr by TSA and TUNEL double staining ( G ) (white arrow indicates TUNEL positive neutrophil, scale bar 10 μM) of mpx: GFP tyrphostin AG825 [Tyr, 10 µM] or CP-724714 [10 µM] treated larvae at three dpf ( H , minimum n = 35 larvae per condition). Uninjured inhibitor treated larvae were assessed for neutrophil apoptosis in the CHT at three dpf ( I , minimum n = 27 larvae per group). Apoptosis at the tail fin injury site of egfra erbb2 crispants at two dpf was also measured at eight hpi ( J , minimum n = 26 larvae per group). All data collated from at least three independent experiments, displayed as mean ± SEM. Each icon shows one data point from one individual larvae. Statistically significant differences were calculated by two-way ANOVA with Sidak post-test ( B–D ) or one-way ANOVA with Dunnett’s post-test(E), Students’ t-test ( F ), Kruskal-Wallis test with Dunn’s post-test ( H–I ) or Mann-Whitney U test ( J ), and indicated as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: C57BL/6 mice (female, 9–10 weeks old) were nebulised with LPS (3 mg per group of 8 mice) ( Pseudomonas aeruginosa , Sigma-Aldrich) and immediately injected intraperitoneally (i.p.) with either Tyrphostin AG825 (Tocris Bioscience) at 20 mg/Kg in 10% DMSO v/v in vegetable oil (eight mice, treatment group) or an equivalent volume of 10% DMSO v/v in vegetable oil (eight mice, control group) ( ; ).

Techniques: Generated, Microscopy, TUNEL Assay, Double Staining, MANN-WHITNEY

Journal: The Journal of clinical endocrinology and metabolism

Article Title: EGF receptor (ERBB1) abundance in adipose tissue is reduced in insulin-resistant and type 2 diabetic women.

doi: 10.1210/jc.2011-1033

Figure Lengend Snippet: FIG. 5. ErbB1, but not ErbB2, tyrosine phosphorylation was increased for at least 6 h after induction of differentiation. ErbB1 and ErbB2 phosphorylation was measured by ELISA (A and B). Total ErbB1 and ErbB2 level was also measured by ELISA (C and D). HB-EGF, TGF, and amphiregulin (AREG) are positive controls (A–D). AG1478 effective concentration was established (E). Various ErbB1 selective (AG1478, erlotinib, and gefitinib), ErbB2 selective (AG825), and ErbB1/ErbB2 dual (lapatinib) inhibitors were compared for their action on ErbB1 phosphorylation (F). Results are expressed as means and SEM of two independent experiments using six pooled individuals (a total of 12 subjects). One-way ANOVA on repeated measurement followed by Bonferroni’s test was used to determine statistical significance. *, P 0.05; **, P 0.01; and ***, P 0.001 compared with time zero (A–D), compared with basal (E and F).

Article Snippet: In the experiments testing the effects of AG1478 (Cell Signaling Technology, Danvers, MA), erlotinib, gefitinib, and lapatinib (LC Laboratories, Woburn, MA), and AG825 (Santa Cruz Biotechnology, Santa Cruz, CA), they were added 30 min before MDI at a concentration of 10 M. A figure showing neutral lipid accumulation can be found in Supplemental Fig. 1, published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org.

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Oncotarget

Article Title: Suppression of tumor angiogenesis by metformin treatment via a mechanism linked to targeting of HER2/HIF-1α/VEGF secretion axis

doi:

Figure Lengend Snippet: MDA-MB-453 or 4T1 cells were cultured with metformin (10 mM), AG825 (10 μM) or HRG-β1 (50 ng/ml) in DMEM containing 10% FBS for 24 h, then the intracellular proteins, mRNA and TCM were extracted. A. Immunoblotting for the total and phosphorylated levels (Tyr 1221/1222) of HER2 and VEGFA proteins in MDA-MB-453 cells. B. Representative images showing the mRNA and secretion levels of VEGF of MDA-MB-453 cells ( n = 5 for both). C. Representative image showing the growth curve of 4T1 tumors from control mice and those treated with metformin (200 mg/kg • day), or AG825 (10 mg/kg. day) ( n = 6 − 8). D. Immunohistochemical staining for VEGFA in 4T1 tumors; scale, 200 μm. E. Immunohistochemical staining for CD31 + vessels and quantitative measurement of microvessel density in 4T1 tumors ( n = 6 − 8). Scale bar, 75 μm. All data is presented as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: AG825 and YC-1 were obtained from Cayman company (USA) and the recombinant human HRG-β1 was purchased from PeproTech corporation (USA).

Techniques: Cell Culture, Western Blot, Control, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Suppression of tumor angiogenesis by metformin treatment via a mechanism linked to targeting of HER2/HIF-1α/VEGF secretion axis

doi:

Figure Lengend Snippet: MDA-MB-453 cells were cultured with metformin (10 mM), AG825 (10 μM), YC-1 (10 μM) or HRG-β1 (50 ng/ml) for 24 h, then the intracellular proteins, mRNA, and tumor cell-conditioned medium (TCM) were extracted. A. YC-1 almost completely inhibited HIF-1α expression of MDA-MB-453 cells even in the presence of HRG-β1 treatment. Representative image showing mRNA levels of B. HIF-1α and C. VEGFA in MDA-MB-453 cells ( n = 5 for both). D. HUVECs were cultured with serum-free medium (SFM) or TCM from MDA-MB-453 cells treated with YC-1 (10 μM), HRG-β1 (50 ng/ml) or both ( n = 6). E. Immunoblotting for protein expression of HIF-1α in MDA-MB-453 cells in both the presence and absence of MG132 (20 μM). F. Immunohistochemical staining for HIF-1α in 4T1 tumors from control mice or those treated with metformin (200 mg/kg • day), YC-1 (10 mg/kg. day), or AG825 (10 mg/kg. day). Red arrows indicate the cells with nuclei positive for HIF-1α. Scale bar: 100 μm. G. Immunofluorescent double staining for CD31 and HIF-1α in 4T1 tumors. Scale bar: 100 μm. All data is presented as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: AG825 and YC-1 were obtained from Cayman company (USA) and the recombinant human HRG-β1 was purchased from PeproTech corporation (USA).

Techniques: Cell Culture, Expressing, Western Blot, Immunohistochemical staining, Staining, Control, Double Staining

Journal: Molecular Cancer

Article Title: Yin Yang-1 suppresses invasion and metastasis of pancreatic ductal adenocarcinoma by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism

doi: 10.1186/1476-4598-13-130

Figure Lengend Snippet: Mechanisms involved in the tumor suppression role of YY1. (a) Effects of YY1 on MUC4, ErbB2, MEF2C, MMP10 and MAPK (Erk1/2, JNK and p38) signaling pathways were assayed by western blotting. BXPC-YY1 indicates YY1overexpressing BXPC-3 cells; BXPC-Vector indicates BXPC-3 cells transfected with a control vector; BXPC-YY1 shRNA indicates YY1 knockdown BXPC-3 cells; BXPC-Scramble shRNA indicates BXPC-3 cells transfected with a vector expressing Scramble shRNA. GAPDH was used as the internal control. (b) Bands were quantified by densitometry. Histograms of the ratio (phosphorylated/constitutive form) for Erk1/2, JNK and p38 MAPK kinases are shown. (c) BXPC-YY1 shRNA cells were transfected with MMP10 siRNA or negative control siRNA. 12 h after transfection, cell migration assays were performed. The upper chambers were seeded with various cell lines. The membranes of the chambers were stained with 0.1% crystal violet. Scale bar, 100 μm. (d) Quantification of the data from Figure c. OD values from three independent experiments were assessed. (e) Luciferase activities of MMP10 promoter-transfected BXPC-Scramble shRNA or BXPC-YY1 shRNA cells treated with MUC4 siRNA or various inhibitors of signal transduction molecules. MUC4 blockage, the ErbB2 inhibitor (AG825, 25 μM) and the p38 inhibitor (SB203580, 10 μM) significantly inhibited YY1 knockdown-stimulated luciferase activity in BXPC-YY1 shRNA cells transfected with the MMP10 promoter. (f) The YY1 knockdown-stimulated luciferase activity could be significantly inhibited by MEF2C blockage. Luciferase activity of MMP10 promoter in BXPC-YY1 shRNA cells was significantly decreased when the presumed MEF2C binding site (nucleotides −881 to −890) was mutated. (*represents p < 0.05, **represents p < 0.01, # represents p < 0.001, when compared to the control group).

Article Snippet: At 12 h after transfection, the medium was replaced and cells were treated for 24 h with 25 μM AG825 (Merck Millipore), 10 μM SB203580 (Cell Signaling Technology) or equivalent amounts of DMSO vehicle (Sigma) as a control.

Techniques: Protein-Protein interactions, Western Blot, Plasmid Preparation, Transfection, Control, shRNA, Knockdown, Expressing, Negative Control, Migration, Staining, Luciferase, Transduction, Activity Assay, Binding Assay