Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: Expression pattern of eyes absent (Eya) 2 in human astrocytoma. (A) Immunohistochemical staining of Eya2 protein in normal brain tissue was negative in glial cells and weak in neurons. The black arrows indicate neurons and the white arrows indicate glial cells. (B) Negative Eya2 staining in pilocytic astrocytoma (grade I). (C) Negative Eya2 staining in grade II diffuse astrocytoma. (D) Weak nuclear Eya2 staining in grade III anaplastic astrocytoma. (E) Strong nuclear Eya2 staining in grade III anaplastic astrocytoma. (F) Strong nuclear Eya2 staining in glioblastoma (grade IV) (magnification, ×200). Scale bar, 50 µ m.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: The association between eyes absent 2 (Eya 2) and the clinical characteristics of astrocytoma.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Expressing

Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: Eyes absent (Eya) 2 expression in astrocytoma cell lines. (A) Western blot analysis indicated that Eya2 and Six1 protein expression was high in the U251 cell line and low in the U87 and A172 cell lines. (B) Eya2 siRNA treatment decreased its mRNA and protein expression in U251 cells. * P<0.05 Eya2 siRNA vs Neg siRNA in U251 cells. Eya2 plasmid transfection upregulated its mRNA and protein levels. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Control

Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: Eyes absent (Eya) 2 regulates the growth and invasion of astrocytoma cells. (A) Cell Counting kit-8 (CCK-8) assay demonstrated that Eya2 overexpression accelerated cell growth rate, while Eya2 knockdown inhibited cell proliferation. (B) Colony formation assay demonstrated that Eya2 overexpression upregulated the colony number, while Eya2 knockdown downregulated the colony number. * P<0.05 Eya2 siRNA vs Neg siRNA in U251 cells. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. (C) Matrigel invasion assay revealed that Eya2 knockdown decreased cell invasion in the U251 cell line. * P<0.05 Eya2 siRNA vs Neg siRNA in U251 cells. Transfection with Eya2 expession plasmid increased the A172 invading cell number. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Cell Counting, CCK-8 Assay, Over Expression, Knockdown, Colony Assay, Plasmid Preparation, Control, Invasion Assay, Transfection

Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: Eyes absent (Eya) 2 regulates cell cycle and related protein expression. (A) Eya2 overexpression in A172 cells increased the S phase percentage and decreased G1 phase percentage. Eya2 knockdown by siRNA in U251 cells had the opposite effect. * P<0.05 Eya2 siRNA vs Neg siRNA in U251 cells. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. (B) Eya2 overexpression upregulated cyclin D1, cyclin E, MMP9 mRNA and protein expression in the A172 cell line. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. Eya2 knockdown downregulated the expression of these proteins in the U251 cells. * P<0.05 Eya2 siRNA vs Neg siRNA in U251 cells. Eya2 overexpression activated ERK phosphorylation, while Eya2 knockdown downregulated ERK phosphorylation.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Expressing, Over Expression, Knockdown, Plasmid Preparation, Control, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: Eyes absent (Eya) 2 interacts with Six1 and regulates ERK signaling. (A) ERK inhibitor, PD98059, blocked the promoting effect of Eya2 on A172 invasion. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. (B) Western blot analysis revealed that ERK inhibitor, PD98059, abolished the promoting effect of Eya2 on MMP9 mRNA and protein expression. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. (C) Eya2 co-immunoprecipitated with Six1 in U251 cells. (D) Transfection with Six1 siRNA significantly downregulated its protein in A172 cells. In Six1-depleted cells, transfection with Eya2 expression plasmid could not upregulate MMP9 expression at both the mRNA and protein level. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. Densitometry of western bands was measured using ImageJ software and relative intensity was calculated and indicated.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Plasmid Preparation, Control, Western Blot, Expressing, Immunoprecipitation, Transfection, Software

Journal: Oncogene

Article Title: Eya2 Is Required to Mediate the Pro-Metastatic Functions of Six1 Via the Induction of TGF-β Signaling, Epithelial-Mesenchymal Transition, and Cancer Stem Cell Properties

doi: 10.1038/onc.2011.259

Figure Lengend Snippet: (a) Eya2 is the most abundant Eya family member in MCF7 cells. Quantitative real time PCR of Eyas1-4, indicating comparative Eya mRNA copy number in MCF7-Six1 or MCF7-Ctrl clones. Error bars represent the standard error of the mean of 3 grouped MCF7-Six1 or MCF7-Ctrl clones from a single RNA isolation. Data is a representative image of triplicate RNA isolations. (b) Real time PCR and (c) immunocytochemistry for Eya2 performed on MCF7 cells stably transfected with Eya2 shRNA or scramble shRNA, and then clonally selected. shRNA-a and shRNA-b are separate clonal isolates containing shRNA constructs targeting different regions of Eya2. Two clonal MCF7-Six1/shRNA isolates from each shRNA group were chosen for analysis. Error bars indicate the standard deviation of the mean of duplicate RNA isolations.

Article Snippet: Primary antibodies included Eya2 (1:300; Ford Lab), Six1 (1:75; Atlas Antibodies, Stockholm, Sweden), and Smad3 (5ug/ml; Invitrogen).

Techniques: Real-time Polymerase Chain Reaction, Clone Assay, Isolation, Immunocytochemistry, Stable Transfection, Transfection, shRNA, Construct, Standard Deviation

Journal: Oncogene

Article Title: Eya2 Is Required to Mediate the Pro-Metastatic Functions of Six1 Via the Induction of TGF-β Signaling, Epithelial-Mesenchymal Transition, and Cancer Stem Cell Properties

doi: 10.1038/onc.2011.259

Figure Lengend Snippet: MCF7-Six1/Eya2 shRNA clones have decreased levels of (a) TβR1 and (b) total Smad3. (c) MCF7-Six1/Eya2 shRNA clones exhibit decreased TGF-β-responsive transcription compared to MCF7-Six1/scrambled controls as observed by performing luciferase activity assays with the 3TP reporter construct. All transfections were normalized to renilla luciferase activity. Data points show the mean of two individual clones and error bars represent the standard error of the mean for 2 experiments. P values represent unpaired t test statistical analysis.

Article Snippet: Primary antibodies included Eya2 (1:300; Ford Lab), Six1 (1:75; Atlas Antibodies, Stockholm, Sweden), and Smad3 (5ug/ml; Invitrogen).

Techniques: shRNA, Clone Assay, Luciferase, Activity Assay, Construct, Transfection

Journal: Oncogene

Article Title: Eya2 Is Required to Mediate the Pro-Metastatic Functions of Six1 Via the Induction of TGF-β Signaling, Epithelial-Mesenchymal Transition, and Cancer Stem Cell Properties

doi: 10.1038/onc.2011.259

Figure Lengend Snippet: Eya2 knockdown in MCF7-Six1 cells reverses the ability of Six1 to (a) upregulate the mesenchymal marker Fibronectin and (b & c) to relocalize E-cadherin and β-catenin from the insoluble (membranous) fraction to the soluble (cytoplasmic) fraction as shown by western blot and quantitation following cell fractionation. (d) MCF7-Six1/Eya2 shRNA clones have decreased β-catenin responsive transcription compared to Six1 scrambled controls. β-catenin transcriptional activity was measured using the TOP-flash luciferase reporter construct normalized to renilla luciferase activity. Data points for fractionation and reporter activity show the mean of two individual clones and error bars represent the standard error of the mean for 2 experiments. P values represent unpaired t test statistical analysis. (e) Eya2 knockdown does not reverse the Six1 induced decrease in cell-matrix adhesion. Relative adherence measured by crystal violet staining.

Article Snippet: Primary antibodies included Eya2 (1:300; Ford Lab), Six1 (1:75; Atlas Antibodies, Stockholm, Sweden), and Smad3 (5ug/ml; Invitrogen).

Techniques: Knockdown, Marker, Western Blot, Quantitation Assay, Cell Fractionation, shRNA, Clone Assay, Activity Assay, Luciferase, Construct, Fractionation, Staining

Journal: Oncogene

Article Title: Eya2 Is Required to Mediate the Pro-Metastatic Functions of Six1 Via the Induction of TGF-β Signaling, Epithelial-Mesenchymal Transition, and Cancer Stem Cell Properties

doi: 10.1038/onc.2011.259

Figure Lengend Snippet: (a) Flow cytometric analysis demonstrates loss of CD44+/CD24− cancer stem cells with knockdown of Eya2. (b) Secondary tumorsphere assays, a measurement of self renewal capacity, demonstrate decreased tumorsphere formation and (c) re-gained spherical shape with knockdown of Eya2. Antibodies used to perform flow cytometry include CD24 and CD44, markers found on human epithelial stem cells. Figure is a representative image of two experiments. Data points show the mean of two individual clones and error bars represent the standard error of the mean. P values represent unpaired t test statistical analysis. Original magnification, x200.

Article Snippet: Primary antibodies included Eya2 (1:300; Ford Lab), Six1 (1:75; Atlas Antibodies, Stockholm, Sweden), and Smad3 (5ug/ml; Invitrogen).

Techniques: Knockdown, Flow Cytometry, Clone Assay

Journal: Oncogene

Article Title: Eya2 Is Required to Mediate the Pro-Metastatic Functions of Six1 Via the Induction of TGF-β Signaling, Epithelial-Mesenchymal Transition, and Cancer Stem Cell Properties

doi: 10.1038/onc.2011.259

Figure Lengend Snippet: (a) Representative images of human breast cancer tissue arrays stained with anti-Six1, anti-Eya2, and anti-Smad3 antibodies show that low Six1 and low Eya2 correlate with little nuclear Smad3, whereas tumors that express both high Six1 and Eya2 show high levels of nuclear Smad3. (b) Quantitation of Smad3 staining. Staining of tissues was scored on a scale from 0-4 for each antibody. Each sample was categorized as having above (high) or below (low) the mean Eya2 and/or Six1 staining and values were plotted against the average Smad3 scores within the group. Statistical analysis performed using Anova. Original magnification, x400. Scale bars: 20um.

Article Snippet: Primary antibodies included Eya2 (1:300; Ford Lab), Six1 (1:75; Atlas Antibodies, Stockholm, Sweden), and Smad3 (5ug/ml; Invitrogen).

Techniques: Staining, Quantitation Assay

Journal: Oncogene

Article Title: Eya2 Is Required to Mediate the Pro-Metastatic Functions of Six1 Via the Induction of TGF-β Signaling, Epithelial-Mesenchymal Transition, and Cancer Stem Cell Properties

doi: 10.1038/onc.2011.259

Figure Lengend Snippet: In a gene expression dataset of 295 women with early-stage invasive breast carcinoma ( van ’t Veer et al ., 2002 ), patient samples expressing high Six1 in the absence of high Eya2, and high Eya2 in the absence of high Six1, do not correlate with reduced time to metastasis, reduced time to relapse, and shortened breast cancer-specific survival while high Six1 and high Eya2 together in the same patient sample significantly correlates with shortened time to relapse and to metastasis, and with shortened breast cancer specific survival. The mean value for Six1 and/or Eya2 expression was used to divide the samples into high (above the mean) and low (below the mean) Six1 and Eya2 expression. P-values were calculated by log-rank analysis.

Article Snippet: Primary antibodies included Eya2 (1:300; Ford Lab), Six1 (1:75; Atlas Antibodies, Stockholm, Sweden), and Smad3 (5ug/ml; Invitrogen).

Techniques: Gene Expression, Expressing

Journal: The Journal of Clinical Investigation

Article Title: A phosphotyrosine switch determines the antitumor activity of ERβ

doi: 10.1172/JCI74085

Figure Lengend Snippet: (A) Co-IP of endogenous EYA2 and ERβ in a substrain of an MCF7 breast cancer cell line that expresses both ER proteins. E2 (10 nM) was used. (B) GST-EYA2 pulldown with in vitro–translated ERβ in the presence of vehicle or various ligands. Five percent input protein was loaded. An ERα-specific agonist, PPT (1 nM), or an ERβ-specific agonist, DPN (10 nM), was used. (C) Real-time RT-PCR assessed the effect of EYA2 overexpression on ERβ-mediated transcription of its target genes MDA7 and pS2 in MCF7 cells. The value for column 1 was set at 1. (D) Effects of EYA2 knockdown on ERβ-mediated transcription of MDA7 and pS2 in MCF7 cells. *P < 0.05; **P < 0.01. Gel images in this and the following figures are representatives of at least 3 independent experiments. Graphs throughout the figures represent the average of at least 3 experiments. Error bars represent SEM.

Article Snippet: Rabbit anti-EYA2 (HPA027024; Sigma-Aldrich), anti–c-ABL (sc-887; Santa Cruz Biotechnology Inc.), anti–total ERβ (68-4; Millipore), and anti–p-Y36 antibodies were used as the primary antibodies for IHC.

Techniques: Co-Immunoprecipitation Assay, In Vitro, Quantitative RT-PCR, Over Expression, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: A phosphotyrosine switch determines the antitumor activity of ERβ

doi: 10.1172/JCI74085

Figure Lengend Snippet: (A) Sequences of mammalian ERα and ERβ orthologs surrounding the Y36 residue of human ERβ. ClustalW was used for the sequence alignment. (B) The anti–p-Y36 antibody recognized WT ERβ, but not the Y36F mutant or ERα, in IP-Western blotting of FLAG-ER proteins from HEK293T cells. (C) IP-Western blot of FLAG-ERβ in HEK293T cells indicated that EYA2 reduced the p-Y36 signal. (D) EYA2 knockdown in HEK293T cells increased the p-Y36 signal of FLAG-ERβ. (E) Ligand-stimulated p-Y36 signal of endogenous ERβ in MDA-MB-231 cells was reduced by EYA2. EYA2-transfected cells were treated with vehicle, E2, or DPN for 2 hours. The lysates were used in an ERβ-specific IP, followed by immunoblotting with the anti–p-Y36 or anti–total ERβ antibody. (F) Recombinant WT EYA2, but not phosphatase-deficient mutant proteins, efficiently dephosphorylated IP FLAG-ERβ in vitro. (G) Real-time RT-PCR compared WT ERβ and the mutants in activation of the ERβ target genes MDA7 and MSMB in MDA-MB-231 cells. Error bars represent SEM. *P < 0.05; **P < 0.01.

Article Snippet: Rabbit anti-EYA2 (HPA027024; Sigma-Aldrich), anti–c-ABL (sc-887; Santa Cruz Biotechnology Inc.), anti–total ERβ (68-4; Millipore), and anti–p-Y36 antibodies were used as the primary antibodies for IHC.

Techniques: Residue, Sequencing, Mutagenesis, Western Blot, Knockdown, Transfection, Recombinant, In Vitro, Quantitative RT-PCR, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: A phosphotyrosine switch determines the antitumor activity of ERβ

doi: 10.1172/JCI74085

Figure Lengend Snippet: (A) WT c-ABL, not a kinase-dead mutant, increased total p-Y and p-Y36–specific signals of FLAG-ERβ in HEK293T cells. Immunoblotting with an anti–c-ABL antibody indicated the physical association between c-ABL and ERβ. (B) p-Y36 of FLAG-ERβ was reduced by imatinib but enhanced by DPH, a c-ABL activator, in HEK293T cells. (C) c-ABL knockdown reduced p-Y36 of endogenous ERβ in MDA-MB-231 cells. (D) WT ERβ was directly phosphorylated in vitro by purified WT, but not mutant, c-ABL. (E) c-ABL knockdown reduced transcription of the ERβ target genes MDA7 and MSMB in MDA-MB-231 cells, which was rescued by an siRNA-resistant c-ABL expression vector. (F) c-ABL knockdown in MDA-MB-231 cells abolished transcriptional activation of MDA7 and MSMB by WT ERβ but not the nonphosphorylatable Y36E mutant. Also included is the Y36F mutant. Error bars represent SEM. *P < 0.05.

Article Snippet: Rabbit anti-EYA2 (HPA027024; Sigma-Aldrich), anti–c-ABL (sc-887; Santa Cruz Biotechnology Inc.), anti–total ERβ (68-4; Millipore), and anti–p-Y36 antibodies were used as the primary antibodies for IHC.

Techniques: Mutagenesis, Western Blot, Knockdown, In Vitro, Purification, Expressing, Plasmid Preparation, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: A phosphotyrosine switch determines the antitumor activity of ERβ

doi: 10.1172/JCI74085

Figure Lengend Snippet: (A) Co-IP in HEK293T cells between FLAG-ERβ proteins and endogenous p300. Shown on the right is normalized quantification of the immunoblots from 3 independent experiments. (B and C) p300 ChIP at the p-Y36–dependent ERβ target promoters MDA7 (B) and MSMB (C), using chromatin from Hs578T cells that expresses either FLAG-tagged WT or mutant Y36F-ERβ. In all ChIP experiments, E2 (10 nM) was added to the estrogen-deprived cells. (D and E) FLAG-ERβ ChIP at the MDA7 (D) and MSMB (E) promoters. (F and G) p300 ChIP at the same ERβ target promoters, with or without EYA2 overexpression. Error bars represent SEM. *P < 0.05; **P < 0.01. EV, empty vector.

Article Snippet: Rabbit anti-EYA2 (HPA027024; Sigma-Aldrich), anti–c-ABL (sc-887; Santa Cruz Biotechnology Inc.), anti–total ERβ (68-4; Millipore), and anti–p-Y36 antibodies were used as the primary antibodies for IHC.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Mutagenesis, Over Expression, Plasmid Preparation

Journal: The Journal of Clinical Investigation

Article Title: A phosphotyrosine switch determines the antitumor activity of ERβ

doi: 10.1172/JCI74085

Figure Lengend Snippet: (A and B) Xenograft tumor growth derived from MDA-MB-231 cells that contained empty vector, WT, or mutant ERβ. The Myc-ERβ proteins and images of individual tumors upon harvest are shown. (C) EYA2 overexpression in MDA-MB-231 cells neutralized the antitumor activity of WT, but not Y36E-mutant, ERβ. (D) Y36E mutant was more resistant to c-ABL knockdown than was WT ERβ in the xenograft tumor model. Error bars represent SEM. *P < 0.05.

Article Snippet: Rabbit anti-EYA2 (HPA027024; Sigma-Aldrich), anti–c-ABL (sc-887; Santa Cruz Biotechnology Inc.), anti–total ERβ (68-4; Millipore), and anti–p-Y36 antibodies were used as the primary antibodies for IHC.

Techniques: Derivative Assay, Plasmid Preparation, Mutagenesis, Over Expression, Activity Assay, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: A phosphotyrosine switch determines the antitumor activity of ERβ

doi: 10.1172/JCI74085

Figure Lengend Snippet: (A) Expression of p-Y36, c-ABL, and EYA2 in human breast cancer tissues. Left panels: Representative IHC staining of p-Y36, c-ABL, and EYA2. Original magnification, ×20; scale bars: 100 εm. (B) Kaplan-Meier estimate of disease-free survival and overall survival in a total of 726 available specimens from the TMA (left graphs) and the stage II and III specimens from the TMA (right graphs), stained with the p-Y36 antibody. Marks on the graph lines represent censored samples. (C) Kaplan-Meier estimate of disease-free survival and overall survival in the total (left graphs) and the stage II and III specimens (right graphs) stained for total ERβ.

Article Snippet: Rabbit anti-EYA2 (HPA027024; Sigma-Aldrich), anti–c-ABL (sc-887; Santa Cruz Biotechnology Inc.), anti–total ERβ (68-4; Millipore), and anti–p-Y36 antibodies were used as the primary antibodies for IHC.

Techniques: Expressing, Immunohistochemistry, Staining