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