Journal: Hypertension

Article Title: Activation of Proteinase-Activated Receptor 2 Stimulates Soluble Vascular Endothelial Growth Factor Receptor 1 Release via Epidermal Growth Factor Receptor Transactivation in Endothelial Cells

doi: 10.1161/hypertensionaha.109.136333

Figure Lengend Snippet: Figure 2. PAR-2–induced sVEGFR-1 release from endothelial cells depends on PKC activity. A, HUVECs and (B) HEK293 cells trans- fected with a VEGFR-1 promoter luciferase reporter construct were pretreated with the PKC inhibitor (Ro-32-0432; 1 mol/L) for 45 minutes and then stimulated with PAR-2 activating peptide (10 mol/L of 2f-LIGRLO-NH2) for 24 hours, and the cell culture superna- tants were assayed for sVEGFR-1 by ELISA (A) and cell lysates assayed for luciferase activity (B), respectively. C, HUVECs were infected overnight with 100 ifu per cell of adenoviruses expressing dominant-negative PKC (dnPKC), PKC (dnPKC), PKC (dnPKC), or empty vector (EV), incubated for 24 hours, and the expression of PKC isoforms were examined in cell lysates by Western blotting. These cells were stimulated with PAR-2–activating peptide (10 mol/L of 2f-LIGRLO-NH2) for 24 hours, and the conditioned medium was assayed for sVEGFR-1 by ELISA. D, PKC-1 siRNA was introduced into HUVECs using an Amaxa nucleofector and the knockdown of PKC1 confirmed by Western blotting. PAR-2–mediated sVEGFR-1 release was inhibited in cells treated with PKC-1 siRNA. Results represent the mean (SEM). *P0.01 (A and B vs control and C vs EV2f-LIGRLO); *P0.05 (D vs control).

Article Snippet: Enzyme-Linked Immunosorbent Assays The sVEGFR-1 concentration in cell supernatants was determined as described previously.5 EGFR was measured in cell lyates using the EGFR DuoSet IC ELISA according to the manufacturer’s instructions (R&D Systems, UK).

Techniques: Activity Assay, Luciferase, Construct, Cell Culture, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Dominant Negative Mutation, Plasmid Preparation, Incubation, Western Blot, Knockdown, Control

Journal: Hypertension

Article Title: Activation of Proteinase-Activated Receptor 2 Stimulates Soluble Vascular Endothelial Growth Factor Receptor 1 Release via Epidermal Growth Factor Receptor Transactivation in Endothelial Cells

doi: 10.1161/hypertensionaha.109.136333

Figure Lengend Snippet: Figure 4. ERK-1/2 activation is required for PAR-2–induced sVEGFR-1 release. A, HUVECs and (B) HEK293 cells transfected with a VEGFR-1 promoter luciferase reporter construct were pretreated with an MEK-1/2 inhibitor (U0126; 10 mol/L) for 45 minutes, stimu- lated with PAR-2–activating peptide (2f-LIGRLO-NH2; 10 mol/L) for 24 hours, and the conditioned medium was assayed for sVEGFR-1 by ELISA (A) and luciferase activity determined in cell lysates (B), respectively. C through G, Western blot analysis: cell lysates were immunoblotted for phosphor–ERK-1/2 (p-ERK), phosphor-Src (pY416Src), and phospho–Raf-1 (pS338Raf-1); HUVECs were either pre- incubated for 45 minutes with (C) MEK-1/2 (U0126; 10 mol/L), (E) Src (PP2; 10 mol/L), and (G) PKC (GF109203X; 5 mol/L) inhibitors or (D and F) infected overnight with adenoviruses expressing dominant-negative PKC (dnPKC), PKC (dnPKC), or PKC (dnPKC) and stimulated with PAR-2–activating peptide (2f-LIGRLO-NH2; 10 mol/L) for 10 minutes. *P0.01 (A) and *P0.05 (B) vs control.

Article Snippet: Enzyme-Linked Immunosorbent Assays The sVEGFR-1 concentration in cell supernatants was determined as described previously.5 EGFR was measured in cell lyates using the EGFR DuoSet IC ELISA according to the manufacturer’s instructions (R&D Systems, UK).

Techniques: Activation Assay, Transfection, Luciferase, Construct, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Incubation, Infection, Expressing, Dominant Negative Mutation, Control

Journal: Hypertension

Article Title: Activation of Proteinase-Activated Receptor 2 Stimulates Soluble Vascular Endothelial Growth Factor Receptor 1 Release via Epidermal Growth Factor Receptor Transactivation in Endothelial Cells

doi: 10.1161/hypertensionaha.109.136333

Figure Lengend Snippet: Figure 5. EGFR transactivation is required for PAR-2–induced sVEGFR-1 release. A, HUVECs were pretreated for 45 minutes with AG1478 (3 mol/L), stimulated for 10 minutes with PAR-2–activating peptides (2f-LIGRLO-NH2; 10 mol/L) or EGF (50 ng/mL), and the level of phosphorylated and total EGFRs were determined in cell lysates (100 g per well) by ELISA (R&D Systems). B, HUVECs were pretreated with AG1478 and stimulated for 24 hours with PAR-2–activating peptide (2f-LIGRLO-NH2), FXa (200 nmol/L), or EGF (50 ng/mL), and sVEGFR-1 was assayed in the cell culture supernatants by ELISA. C, After pretreatment with the EGFR inhibitor (AG1478), HUVECs were incubated with 2f-LIGRLO-NH2 and FXa (200 nmol/L) for 10 minutes, and cell lysates (30 g per lane) were subjected to Western blotting for phosphor-ERK1/2 (p-ERK) and -actin as a loading control. D, HUVECs were incubated with the Src inhibitor PP2 for 45 minutes and then stimulated for 10 minutes with 2f-LIGRLO-NH2, and the levels of EGFR and phosphorylated EGFR were deter- mined in cell lysates (100 g per well) by ELISA. E, HUVECs were treated with AG1478 before incubation with 2f-LIGRLO-NH2 for 10 minutes, and cell lysates were Western blotted for phosphorylated-Src (p-Src). F, HUVECs were preincubated with the metalloprotein- ase inhibitor (GM6001; 10 mol/L) or control (rGM6001; 10 mol/L) for 45 minutes before stimulation with 2f-LIGRLO-NH2 or FXa (200 nmol/L), and sVEGFR-1 was assayed in cell supernatants by ELISA. Results represent the mean (SEM); n3. *P0.05 (A and D) vs control.

Article Snippet: Enzyme-Linked Immunosorbent Assays The sVEGFR-1 concentration in cell supernatants was determined as described previously.5 EGFR was measured in cell lyates using the EGFR DuoSet IC ELISA according to the manufacturer’s instructions (R&D Systems, UK).

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

Journal: Hypertension

Article Title: Activation of Proteinase-Activated Receptor 2 Stimulates Soluble Vascular Endothelial Growth Factor Receptor 1 Release via Epidermal Growth Factor Receptor Transactivation in Endothelial Cells

doi: 10.1161/hypertensionaha.109.136333

Figure Lengend Snippet: Figure 6. PAR-2-induced sVEGFR-1 release is inhibited by statins, HO-1, and CO. A, HUVECs were stimulated with PAR-2–activating peptide (2f-LIGRLO-NH2; 10 mol/L) or FXa (200 nmol/L) in the presence or absence of Simvastatin (10 mol/L) for 24 hours, and sVEGFR-1 was quantified in cell supernatants by ELISA. HO-1 upregulation in HUVECs incubated with Simvastatin (10 mol/L) for 24 hours was confirmed by Western blotting (inset). B, PAEC-PAR-2 and (C) HEK-293 cells were transfected with the VEGFR-1 promoter- reporter construct, stimulated with PAR-2–activating peptide (2f-LIGRLO-NH2) for 24 hours in the presence or absence of Simvastatin (10 mol/L), and luciferase activity was determined in cell lysates. D, HUVECs were then infected with 50 ifu per cell of HO-1 or control (EV) adenovirus for 48 hours before stimulation with 2f-LIGRLO-NH2 for 24 hours (*P0.001 vs control). HUVECs infected with low (5 ifu per cell; lanes 2 and 4) or high (50 ifu per cell; lanes 3 and 5) concentrations of HO-1 adenovirus for 24 (lanes 2 and 3) or 48 (lanes 4 and 5) hours or uninfected control cells (lane 1) were immunoblotted for HO-1 (inset). E, HEK293 cells containing the VEGFR-1 promoter-reporter construct were electroporated with HO-1 siRNA and incubated overnight. After stimulation with 2f-LIGRLO-NH2 (10 mol/L) for 24 hours, luciferase activity was determined in cell lysates. The medium was then collected and assayed for sVEGFR-1 by ELISA. F, HUVECs were treated with 50 mol/L of CORM-2 or CORM-2 control for 45 minutes and then stimulated for 24 hours with 2f-LIGRLO-NH2. Results represents mean (SEM); n3. A through C, *P0.01 vs control; D, *P0.001 vs control; **P0.001 vs EV2f-LIGRLO; E, *P0.05, *P0.001, and **P0.05 vs control.

Article Snippet: Enzyme-Linked Immunosorbent Assays The sVEGFR-1 concentration in cell supernatants was determined as described previously.5 EGFR was measured in cell lyates using the EGFR DuoSet IC ELISA according to the manufacturer’s instructions (R&D Systems, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Transfection, Construct, Luciferase, Activity Assay, Infection, Control

Journal: Breast cancer research : BCR

Article Title: Heregulin β1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells

doi: 10.1186/bcr1754

Figure Lengend Snippet: Clinicopathological parameters for the clinical tumour set

Article Snippet: The antibodies used were total EGFR (SC-03) erbB2 (SC-284), erbB3 (SC-285) and erbB4 (SC-283) (Insight Biotechnology Ltd), anti-phospho-erbB2 (pY1248, 2247), anti-phospho-EGFR (pY1068, 2234), total AKT (9272), phospho-AKT (pS473, 9271), total ERK1/2 (9102) and phospho-ERK1/2 (pT202/pY204, 9101) (New England Biolabs, Hitchin, Hertfordshire, UK), and β-actin (AC-15) (Sigma).

Techniques:

Journal: Breast cancer research : BCR

Article Title: Heregulin β1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells

doi: 10.1186/bcr1754

Figure Lengend Snippet: Effects of gefitinib on erbB receptor dimerization patterns and activity of associated downstream signalling elements. (a) Western blot (WB) analysis of phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated erbB2, phosphorylated tyrosine (Tyr) and total erbB3 protein expression following immunoprecipitation with total erbB3 antibody in tamoxifen-resistant MCF-7 (Tam-R) cells prior to and following incubation of Tam-R cells with gefitinib (1 μM) for 1 hour. (b) WB analysis of total and phosphorylated EGFR, erbB2, AKT and ERK1/2 protein expression in Tam-R cells prior to and following incubation with gefitinib (1 μM) for 1 hour. (c) WB analysis of total and phosphorylated EGFR, erbB2 and erbB3 protein expression following immunoprecipitation with total EGFR antibody in Tam-R cells prior to and following incubation with gefitinib (1 μM) for 1 hour. Tamoxifen (100 nM) was present in all studies. Data are representative of at least three separate experiments.

Article Snippet: The antibodies used were total EGFR (SC-03) erbB2 (SC-284), erbB3 (SC-285) and erbB4 (SC-283) (Insight Biotechnology Ltd), anti-phospho-erbB2 (pY1248, 2247), anti-phospho-EGFR (pY1068, 2234), total AKT (9272), phospho-AKT (pS473, 9271), total ERK1/2 (9102) and phospho-ERK1/2 (pT202/pY204, 9101) (New England Biolabs, Hitchin, Hertfordshire, UK), and β-actin (AC-15) (Sigma).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Incubation

Journal: Breast cancer research : BCR

Article Title: Heregulin β1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells

doi: 10.1186/bcr1754

Figure Lengend Snippet: Effects of HRGβ1 and gefitinib on erbB receptor dimerization patterns and associated downstream signalling activity. (a) Western blot (WB) analysis of phosphorylated epidermal growth factor receptor (EGFR), phosphorylated erbB2, phosphorylated tyrosine (Tyr) and total erbB3 protein expression following immunoprecipitation with total erbB3 antibody in tamoxifen-resistant MCF-7 (Tam-R) cells prior to and following treatment with either gefitinib (1 μM) or vehicle control for 1 hour followed by HRGβ1 (10 ng/ml) for 5 minutes. (b) WB analysis of total and phosphorylated EGFR, erbB2, AKT and ERK1/2 protein expression in Tam-R cells prior to and following incubation with either gefitinib (1 μM) or vehicle control for 1 hour followed by either HRGβ1 (10 ng/ml) or vehicle control for 5 minutes. Tamoxifen was also present in all studies. Data are representative of at least three separate experiments.

Article Snippet: The antibodies used were total EGFR (SC-03) erbB2 (SC-284), erbB3 (SC-285) and erbB4 (SC-283) (Insight Biotechnology Ltd), anti-phospho-erbB2 (pY1248, 2247), anti-phospho-EGFR (pY1068, 2234), total AKT (9272), phospho-AKT (pS473, 9271), total ERK1/2 (9102) and phospho-ERK1/2 (pT202/pY204, 9101) (New England Biolabs, Hitchin, Hertfordshire, UK), and β-actin (AC-15) (Sigma).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Incubation

Journal: Breast cancer research : BCR

Article Title: Heregulin β1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells

doi: 10.1186/bcr1754

Figure Lengend Snippet: Effects of combining gefitinib with trastuzumab or LY294002 on HRGβ1-driven signalling in tamoxifen-resistant MCF-7 cells. Western analysis of total and phosphorylated epidermal growth factor receptor (EGFR), erbB2, AKT and ERK1/2 protein expression in tamoxifen-resistant MCF-7 (Tam-R) cells prior to and following incubation with either (a) trastuzumab (100 nM) or vehicle control for 7 days, (b) LY294002 (10 μM) or vehicle control for 1 hour, (c) gefitinib (1 μM), gefitinib in combination with trastuzumab or vehicle control for 7 days, and (d) gefitinib, gefitinib in combination with LY294002 or vehicle control for 1 hour, all followed by either HRGβ1 (10 ng/ml) or vehicle control for 5 minutes. Tamoxifen was also present in all studies. Data are representative of at least three separate experiments.

Article Snippet: The antibodies used were total EGFR (SC-03) erbB2 (SC-284), erbB3 (SC-285) and erbB4 (SC-283) (Insight Biotechnology Ltd), anti-phospho-erbB2 (pY1248, 2247), anti-phospho-EGFR (pY1068, 2234), total AKT (9272), phospho-AKT (pS473, 9271), total ERK1/2 (9102) and phospho-ERK1/2 (pT202/pY204, 9101) (New England Biolabs, Hitchin, Hertfordshire, UK), and β-actin (AC-15) (Sigma).

Techniques: Western Blot, Expressing, Incubation

Journal: Breast cancer research : BCR

Article Title: Heregulin β1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells

doi: 10.1186/bcr1754

Figure Lengend Snippet: Immunohistochemical staining for HRGβ1 in primary breast cancer specimens. (a) Examples of high and low HRGβ1 expression. Scale bars = 20 μm. (b) Box-plots illustrating cytoplasmic HRGβ1 immunohistochemistry assessed by H-score analysis in membrane (mem) epidermal growth factor receptor (EGFR)-negative, erbB2-negative and erbB3-negative primary breast cancer versus membrane EGFR-positive, erbB2-positive and erbB3-positive primary breast cancer. A significant positive correlation between cytoplasmic HRGβ1 expression and membrane erbB receptor positivity was only seen with EGFR (Mann–Whitney U test, P = 0.04). (c) Box-plots illustrating cytoplasmic HRGβ1 immunohistochemistry assessed by H-score analysis in membrane (mem) phosphorylated erbB2-negative versus membrane phosphorylated erbB2-positive primary breast cancer and in nuclear (nuc) phosphorylated ERK1/2-negative versus nuclear phosphorylated ERK1/2-positive primary breast cancer (Mann–Whitney U test, P = 0.006 and P = 0.017, respectively), and scatter plot illustrating the significant positive correlation between expression levels of nuclear phosphorylated AKT and cytoplasmic HRGβ1 in the same samples (Spearman rank test, P = 0.044).

Article Snippet: The antibodies used were total EGFR (SC-03) erbB2 (SC-284), erbB3 (SC-285) and erbB4 (SC-283) (Insight Biotechnology Ltd), anti-phospho-erbB2 (pY1248, 2247), anti-phospho-EGFR (pY1068, 2234), total AKT (9272), phospho-AKT (pS473, 9271), total ERK1/2 (9102) and phospho-ERK1/2 (pT202/pY204, 9101) (New England Biolabs, Hitchin, Hertfordshire, UK), and β-actin (AC-15) (Sigma).

Techniques: Immunohistochemical staining, Staining, Expressing, Immunohistochemistry, MANN-WHITNEY

Journal: Brain Pathology

Article Title: Congenital Glioblastoma: A Clinicopathologic and Genetic Analysis

doi: 10.1111/j.1750-3639.2007.00071.x

Figure Lengend Snippet: Genetic and immunohistologic markers in congenital glioblastoma. Abbreviations: NI = non‐informative; NA = not available; wt = wild type; GBM = glioblastoma.

Article Snippet: Slides were then incubated at room temperature with antibodies directed toward glial fibrillary acidic protein (GFAP, monoclonal, 1:100, Dako Co., Carpinteria, CA, USA), synaptophysin (monoclonal, 1:50; Boehringer Mannheim, Mannheim, Germany), MIB‐1 (monoclonal, 1:150, Immunotech, Inc., Westbrook, ME, USA), EGFR (monoclonal, 1:200 clone 528; Oncogene Science, Cambridge, MA) and p53 (mouse monoclonal, 1:20; DO‐7 DAKO Co., Carpinteria, CA).

Techniques:

Journal: Brain Pathology

Article Title: Congenital Glioblastoma: A Clinicopathologic and Genetic Analysis

doi: 10.1111/j.1750-3639.2007.00071.x

Figure Lengend Snippet: p53 and EGFR expression in congenital glioblastoma. A. Immunohistochemistry for p53 showing strong (3+) nuclear immunoreactivity (Case 5). B. Immunostains for EGFR of (case 4) demonstrated mild (1+) reactivity focally.

Article Snippet: Slides were then incubated at room temperature with antibodies directed toward glial fibrillary acidic protein (GFAP, monoclonal, 1:100, Dako Co., Carpinteria, CA, USA), synaptophysin (monoclonal, 1:50; Boehringer Mannheim, Mannheim, Germany), MIB‐1 (monoclonal, 1:150, Immunotech, Inc., Westbrook, ME, USA), EGFR (monoclonal, 1:200 clone 528; Oncogene Science, Cambridge, MA) and p53 (mouse monoclonal, 1:20; DO‐7 DAKO Co., Carpinteria, CA).

Techniques: Expressing, Immunohistochemistry

Journal: Oncology Letters

Article Title: Increased MIR31HG lncRNA expression increases gefitinib resistance in non-small cell lung cancer cell lines through the EGFR/PI3K/AKT signaling pathway

doi: 10.3892/ol.2017.5878

Figure Lengend Snippet: Knockdown of MIR31HG alters protein expression and cell cycle distribution in PC9-R cells. (A) Western blot analysis revealed that PC9-R cells transfected with si-MIR31HG repressed p-EGFR, p-PI3K, p-AKT and p-Mdm-2 expression, but did not alter total EGFR, PI3K or AKT levels. It also stimulated expression of p53. (B) The result showed that PC9-R cells containing si-MIR31HG increased expression of the proteins Caspase-3, Caspase-9 and Bax, but repressed Bcl-2, compared to levels in the control group. (C) The effect of si-MIR31HG on cell cycle was analyzed by flow cytometry. This showed that PC9-R cells transfected with si-MIR31HG were able to arrest the cell cycle at the G0/G1 phase. EGFR, epidermal growth factor receptor; PI3K, phosphatidylinositol-3 kinase; AKT, protein kinase B; p-EGFR, phosphorylated epidermal growth factor receptor; p-I3K, phosphorylated phosphatidylinositol-3 kinase; p-AKT, phosphorylated protein kinase B; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and incubated with antibodies against p-EGFR (cat. no. PL-0302648; dilution, 1/1,000; PLLABS, Nanaimo, BC, Canada), total EGFR (cat. no. AB36836; dilution, 1:2,000; AbSci, Vancouver, BC, Canada), p-PI3K (cat. no. BS4605; dilution, 1:2,000; Bioworld Technology, Inc., St. Louis Park, MN, USA), total PI3K (cat. no. NB100-75198; dilution, 1:1,500; Novus Biologicals, Inc., Abingdon, UK), p-AKT (cat. no. xyP001a; dilution, 1:3,000; Abcam, Cambridge, UK), total AKT (cat. no. AB27174; dilution, 1:10,000; AbSci), phosphorylated mouse minute 2 homolog (p-Mdm2; cat. no. US1506136; dilution, 1:500; Merck & Co., Inc., Whitehouse Station, NJ, USA), P53 (cat. no. 1026-1; dilution, 1:2,000: Epitomics, Burlingame, CA, USA), GAPDH (cat. no. PA116777; dilution, 1:2,000: Thermo Fisher Scientific, Inc.), Caspase-3 (cat. no. 1087-1; dilution, 1:1,000; Epitomics), Caspase-9 (cat. no. DB081; dilution, 1:5,000; Acris Antibodies GmbH; OriGene, Herford, Germany), Bax (cat. no. PA112602; dilution, 1:1,000; Thermo Fisher Scientific, Inc.) and Bcl-2 (cat. no. MA126233; dilution, 1:1,000; Thermo Fisher Scientific, Inc.) at 4°C overnight.

Techniques: Expressing, Western Blot, Transfection, Flow Cytometry