Journal: Cell Death & Disease

Article Title: Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer

doi: 10.1038/cddis.2016.346

Figure Lengend Snippet: EHF is identified as a new HER2 transcription factor and the modulator of HER3 and HER4 in gastric cancer. ( a ) BGC823 cells were transiently transfected with pGL3-Basic or luciferase reporter constructs containing various lengths of the promoter region of HER2 gene, as indicated (F1: −607/+11; F2: −175/+11; F3: −607/−175) (left panels). Cotransfection with empty vector was used as a control. The ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays (right panels). ( b ) The luciferase reporter gene assay was performed to evaluate the effect of EHF knockdown on promoter activity of HER2 in BGC823 cells. The ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays. ( c ) HEK293T cells were cotransfected pGL3-HER2-Luc-F1 and various amounts of pcDNA3.1(-)A-EHF or empty vector, respectively. Promoter activities of HER2 were measured by luciferase reporter gene assays. All the ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays. ( d ) Putative promoter regions of HER2 (−607/+11), HER3 (−997/+440) and HER4 (−697/+306) were inserted into the pGL3-Basic to construct the luciferase reporter plasmid pGL3-HER2-Luc, pGL3-HER3-Luc and pGL3-HER4-Luc (upper panels). P1-P7 represent the regions analyzed by ChIP assays for HER2 , HER3 and HER4 , respectively. BGC823 cells were transiently transfected with pcDNA3.1/myc-His(-)A-EHF or empty vector, and were subjected to ChIP-qRT-PCR assays using anti-Myc tag antibody. Flod enrichment was shown as means±S.E. of three independent assays (lower panels). ( e ) EMSA assay was performed to confirm the interaction between EHF and HER2 promoter. Shown are specific DNA-binding of in vitro translated EHF protein to an oligonucleotide sequence (SH2) containing ETS responsive element (GAGGAA) from the HER2 promoter. Unlabeled mutated probes contain specific mutations in the GGAA ETS core or flanking nucleotides of core sequence, as indicated by MT1 and MT2. Unlabeled wild-type (WT) and mutated (MT1 or MT2) competitor probes were added at 100-fold molar excess. Statistically significant differences were indicated: * P <0.05; ** P <0.01; *** P <0.001

Article Snippet: The membranes were blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20) and incubated with the indicated primary antibodies, including anti-EHF (Abcam, Inc), anti-total-Erk1/2 (Abcam, Inc), anti-phospho-Erk1/2 (Epitomics, Inc), anti-phospho-AktSer473 (Bioworld Technology, co, Ltd), anti-total-Akt (Bioworld Technology, co, Ltd), anti-HER2 (Sino Biological, Inc), anti-HER3 (Sino Biological, Inc), anti-HER4 (Sino Biological, Inc), anti-E-cadherin (Epitomics, Inc), anti-Vimentin (Epitomics, Inc) and anti-GAPDH (Abgent, Inc).

Techniques: Transfection, Luciferase, Construct, Cotransfection, Plasmid Preparation, Activity Assay, Reporter Gene Assay, Quantitative RT-PCR, Binding Assay, In Vitro, Sequencing

Journal: Cancer control : journal of the Moffitt Cancer Center

Article Title: ERBB4 Expression in Ovarian Serous Carcinoma Resistant to Platinum-Based Therapy

doi: 10.1177/107327481702400115

Figure Lengend Snippet: Mean level of ERBB4 expression in normal controls, tumor tissue, and ovarian cancer cell lines. Analysis of variance was statistically significant (P < .001). The mean scores of ERBB4 are significantly different for tumors vs controls vs cell lines, with cell lines greater than tumors and greater than controls.

Article Snippet: A mouse monoclonal antibody that reacts to ERBB4, NB100–2662 (Novus, Littleton, CO), was used at a 1:25 concentration in Antibody Diluent (Dako, Carpenteria, CA) and incubated for 60 minutes.

Techniques: Expressing

Journal: Cancer control : journal of the Moffitt Cancer Center

Article Title: ERBB4 Expression in Ovarian Serous Carcinoma Resistant to Platinum-Based Therapy

doi: 10.1177/107327481702400115

Figure Lengend Snippet: ERBB4 expression by immunohistochemistry. Shown are (A) rare membranous, (B) nuclear staining, (C–G) increasing H scores from less than 50 to 300 for particular fields, and (H) ERBB4 staining in cells lines.

Article Snippet: A mouse monoclonal antibody that reacts to ERBB4, NB100–2662 (Novus, Littleton, CO), was used at a 1:25 concentration in Antibody Diluent (Dako, Carpenteria, CA) and incubated for 60 minutes.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Cancer control : journal of the Moffitt Cancer Center

Article Title: ERBB4 Expression in Ovarian Serous Carcinoma Resistant to Platinum-Based Therapy

doi: 10.1177/107327481702400115

Figure Lengend Snippet: Using an unpaired t test with Welch correction, ERBB4 expression was higher patients with an IR than CR to platinum-based therapy (P = .033).

Article Snippet: A mouse monoclonal antibody that reacts to ERBB4, NB100–2662 (Novus, Littleton, CO), was used at a 1:25 concentration in Antibody Diluent (Dako, Carpenteria, CA) and incubated for 60 minutes.

Techniques: Expressing

Journal: Cancer control : journal of the Moffitt Cancer Center

Article Title: ERBB4 Expression in Ovarian Serous Carcinoma Resistant to Platinum-Based Therapy

doi: 10.1177/107327481702400115

Figure Lengend Snippet: Multivariate Survival Analysis Using a Cox Proportional Hazard Model

Article Snippet: A mouse monoclonal antibody that reacts to ERBB4, NB100–2662 (Novus, Littleton, CO), was used at a 1:25 concentration in Antibody Diluent (Dako, Carpenteria, CA) and incubated for 60 minutes.

Techniques:

Journal: Cancer control : journal of the Moffitt Cancer Center

Article Title: ERBB4 Expression in Ovarian Serous Carcinoma Resistant to Platinum-Based Therapy

doi: 10.1177/107327481702400115

Figure Lengend Snippet: Log-rank test results illustrating ERBB4 expression and OS. Probability values were as follows: all 3 levels, P = .020; low vs intermediate, P = .080; low vs high, P = .004; and intermediate vs high, P = .201.

Article Snippet: A mouse monoclonal antibody that reacts to ERBB4, NB100–2662 (Novus, Littleton, CO), was used at a 1:25 concentration in Antibody Diluent (Dako, Carpenteria, CA) and incubated for 60 minutes.

Techniques: Expressing