Journal: PLoS ONE

Article Title: Ancestry as a potential modifier of gene expression in breast tumors from Colombian women

doi: 10.1371/journal.pone.0183179

Figure Lengend Snippet: Differentially expressed genes for Luminal B vs. Luminal A tumors according to European ancestry group.

Article Snippet: TaqMan probes were used to quantify the levels of mRNA expression of candidate genes: ERBB2 (Hs01001580_m1), GRB7 (Hs00917999_g1), ONECUT2 (Hs00191477_m1).

Techniques:

Journal: PLoS ONE

Article Title: Ancestry as a potential modifier of gene expression in breast tumors from Colombian women

doi: 10.1371/journal.pone.0183179

Figure Lengend Snippet: Differentially expressed genes in Luminal B tumors according to the Indigenous American ancestry groups.

Article Snippet: TaqMan probes were used to quantify the levels of mRNA expression of candidate genes: ERBB2 (Hs01001580_m1), GRB7 (Hs00917999_g1), ONECUT2 (Hs00191477_m1).

Techniques:

Journal: PLoS ONE

Article Title: Ancestry as a potential modifier of gene expression in breast tumors from Colombian women

doi: 10.1371/journal.pone.0183179

Figure Lengend Snippet: Association between candidate genes expression and the interaction between Indigenous American ancestry and intrinsic subtype.

Article Snippet: TaqMan probes were used to quantify the levels of mRNA expression of candidate genes: ERBB2 (Hs01001580_m1), GRB7 (Hs00917999_g1), ONECUT2 (Hs00191477_m1).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Ancestry as a potential modifier of gene expression in breast tumors from Colombian women

doi: 10.1371/journal.pone.0183179

Figure Lengend Snippet: Association between candidate genes expression and the interaction between European ancestry and intrinsic subtype.

Article Snippet: TaqMan probes were used to quantify the levels of mRNA expression of candidate genes: ERBB2 (Hs01001580_m1), GRB7 (Hs00917999_g1), ONECUT2 (Hs00191477_m1).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Astragalus propinquus schischkin and Salvia miltiorrhiza bunge promote angiogenesis to treat myocardial ischemia via Ang-1/Tie-2/FAK pathway

doi: 10.3389/fphar.2022.1103557

Figure Lengend Snippet: AS regulates Ang-1/Tie-2/FAk pathway. (A) Protein level of p-Tie-2, Ang-1 and Ang-2, results are expressed as mean ± SD, n = 3, ** p < .01, * p < .05. (B) Quantitative analysis of Ang-1, Ang-2 and p-Tie-2 concentration, n = 3. Results are expressed as mean ± SD, ** p < .001, * p < .05, ns indicates not significant. (C) Protein level of FAK and GRB7, results are expressed as mean ± SD, n = 3, ** p < .01, * p < .05. (D) IF staining, the green region is FAK, results are expressed as mean ± SD, n = 3, ** p < .01, * p < .05.

Article Snippet: After blocking with 5% fat-free dry milk in Tris-buffered saline containing .1% Tween-20 for 2 h, the membranes were incubated with the following primary antibodies overnight at 4°C: Collagen 1 (1:1000, #72026, Cell Signaling), matrix metalloproteinase-9 (MMP9) (1:1000, 10375-2-AP, Proteintech), PDGFR-β (1:1000, 13449-1-ap, Proteintech), NG2 (1:1000, #4226, Cell Signaling), VE-cadherin (1:1000, ab33168, Abcam), VEGF (1:1000, 19003-1-ap, Proteintech), TGF-β (1:1000, 21898-1-ap, Proteintech), Ang-1 (1:1000, ab183701, Abcam), Ang-2 (1:1000, ab155106, Abcam), phospho-Tie-2 (1:1000, #4226, Cell Signaling), GRB7 (1:1000, 10045-1-Ig, Proteintech), FAK (1:1000, ab40794, Abcam) and β-actin (1:1000, 66009-1-lg, Proteintech).

Techniques: Concentration Assay, Staining

Journal: Theranostics

Article Title: Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness by targeting GRB7 and MAPK/ERK pathways

doi: 10.7150/thno.22377

Figure Lengend Snippet: The expression of miR-193a-3p is downregulated and inversely correlates with GRB7 expression in ovarian cancers. (A) Comparison of GRB7 expression in human ovarian cancer with other tumor types using the cBioPortal database. The dots represent clinical cases, and the line in the upper and lower box plot represents the upper and lower quartile of the relative mRNA levels of all samples. (B) The Venn diagram shows miR-193a-3p and miR-193b-3p commonly predicted by miRDB, TargetScan, and Miranda. (C) A schematic diagram showing the same binding site of miR-193a-3p and miR-193b-3p at position 332-338 of human GRB7 3' UTR. (D) Western blot analysis of GRB7 expression in ovarian cancer cell lines and HOSEs. (E) qPCR analysis of miR-193a-3p ( upper ) and miR-193b-3p ( lower ) expression in ovarian cancer cell lines. SNORD48 was used as an internal control. (F) Western blot analysis showing that transient transfection of pmR-ZsGreen1-miR-193a-3p or pmR-ZsGreen1-miR-193b-3p reduced the expression of GRB7 in SKOV3 or OVCA433 cells in a dose-dependent manner. (G) A schematic diagram showing the putative miR-193a-3p binding site in wild-type and mutant GRB7 3'UTR ( Upper ). Luciferase reporter assay showing the relative luciferase activity of wild-type or mutant GRB7 3'UTR targeted by a miR-193a-3p using pmR-ZsGreen1-miR-193a-3p plasmid in HEK293 cells ( Lower ).

Article Snippet: Ovarian cancer cells with stable GRB7 knockdown were infected with lentiviral particles carrying GRB7 shRNA plasmid (Santa Cruz).

Techniques: Expressing, Comparison, Binding Assay, Western Blot, Control, Transfection, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation

Journal: Theranostics

Article Title: Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness by targeting GRB7 and MAPK/ERK pathways

doi: 10.7150/thno.22377

Figure Lengend Snippet: MiR-193a-3p targets not only GRB7, but other key factors along MAPK/ERK signaling pathway in ovarian cancer cells. (A) The Venn diagram displays 77 common putative targets of miR-193a-3p predicted from the three algorithms, miRDB, miRanda and TargetScan Human 5.2, including three putative targets, ERBB4, KRAS and SOS2, involved in the MAPK/ERK signaling pathway. (B) Functional annotation analysis using the DAVID functional annotation tool ( http://david.abcc.ncifcrf.gov/ ) showing the main signaling pathways associated with these 78 common targets of miR-193a-3p . (C) Duolink proximity ligation assay (PLA) with fluorescence and confocal microscopy showing the interactions between GRB7 and SOS2 and between GRB7 and KRAS (red dots) by transient transfection of GRB7-expressing plasmid ( Upper ). Anti-Myc, anti-SOS2 and anti-KRAS were used to detect Myc-tagged GRB7 and endogenous SOS2 and KRAS. Transient transfection of HA-SOS2 also showing the interaction between SOS2 and KRAS (red dots) using PLA and anti-HA as well as anti-KRAS ( Lower ). Scale bar, 20 μm. n = 3 independent experiments. (D) Relative luciferase activity of luciferase reporters with wild-type ERBB4, SOS2 and KRAS 3'UTRs co-transfected with miR-193a-3p . (E) Western blot analysis showing that transient transfection of GFP/GRB7-expressing plasmid profoundly elevated ERK activity in HEK293, SKOV3 and OVCA433 cells. (F) Western blot analysis indicating that transient transfection of pmR-ZsGreen1-miR-193a-3p reduced the expression of GRB7, ERBB4, SOS2, KRAS and pERK1/2 in SKOV3 cells. Cell lysates of all the above cell transfectants were harvested 24 h after cell transfection.

Article Snippet: Ovarian cancer cells with stable GRB7 knockdown were infected with lentiviral particles carrying GRB7 shRNA plasmid (Santa Cruz).

Techniques: Functional Assay, Protein-Protein interactions, Proximity Ligation Assay, Fluorescence, Confocal Microscopy, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Western Blot

Journal: Theranostics

Article Title: Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness by targeting GRB7 and MAPK/ERK pathways

doi: 10.7150/thno.22377

Figure Lengend Snippet: DNA methylation leads to downregulation of miR-193a-3p during the tumor development and progression of ovarian cancer cells. (A) qPCR analysis revealing a stepwise decrease in miR-193a-3p expression from Stages 1 to 4 and Grades 1 to 3 in ovarian cancers. (B) Representative photographs showing an inverse correlation between miR-193a-3p and GRB7 expression by ISH and IHC analyses for serous subtype ovarian cancer samples (early stage = Stage 1 and Grade 2, and advanced stage = Stage 2 and Grade 3) on a commercial ovarian cancer tissue array (OVC1021, Pantomics). Scale bar, 100 μm. (C) The expression of miR-193a-3p could be restored in ovarian cancer cells upon treatment with 5-Aza-dc (5 μM) for 4 days. (D) Verification of miR-193a-3p promoter methylation by MS-PCR in ovarian cancer cells upon 5-Aza-dC treatment. Treatment with 5-Aza-dC (5 μM) for 3-4 days. (E) Pyrosequencing analysis showing a progressive increase in DNA methylation with the tumor stage ( Upper ) and grade ( Lower ) of ovarian cancers.

Article Snippet: Ovarian cancer cells with stable GRB7 knockdown were infected with lentiviral particles carrying GRB7 shRNA plasmid (Santa Cruz).

Techniques: DNA Methylation Assay, Expressing, Methylation

Journal: Theranostics

Article Title: Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness by targeting GRB7 and MAPK/ERK pathways

doi: 10.7150/thno.22377

Figure Lengend Snippet: Clinico-pathological correlation of the expression of miR193a-3p in an ovarian cancer tissue array (OVC1021). The 4-fold and 3-fold cut-off points of miR-193a-3p and GRB7, respectively, were determined by ROC analysis.

Article Snippet: Ovarian cancer cells with stable GRB7 knockdown were infected with lentiviral particles carrying GRB7 shRNA plasmid (Santa Cruz).

Techniques: Expressing

Journal: Theranostics

Article Title: Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness by targeting GRB7 and MAPK/ERK pathways

doi: 10.7150/thno.22377

Figure Lengend Snippet: Re-introduction of miR-193a-3p suppresses cell proliferation, cell migration and invasion capacities of ovarian cancer cells. (A) qPCR and Western blot analyses of miR193a-3p and GRB7 expression in stable miR-193a-3p-expressing SKOV3 and OVCA433 cells. SNORD48 was used as an internal control. (B) XTT proliferation assay, (C) Transwell cell migration and invasion assays showing that stable re-expression of miR-193a-3p inhibited cell proliferation rates, cell migration and invasion abilities of SKOV3 and OVCA433 ovarian cancer cells.

Article Snippet: Ovarian cancer cells with stable GRB7 knockdown were infected with lentiviral particles carrying GRB7 shRNA plasmid (Santa Cruz).

Techniques: Migration, Western Blot, Expressing, Control, Proliferation Assay

Journal: Theranostics

Article Title: Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness by targeting GRB7 and MAPK/ERK pathways

doi: 10.7150/thno.22377

Figure Lengend Snippet: The functional role of GRB7 knockdown on cell proliferation, migration, invasion, and in vivo tumorigenicity. (A) Western blot analysis showing the levels of GRB7 knockdown (KD) and scrambled control (SCR) in SKOV3 cells. (B) XTT proliferation assay and (C) Transwell cell migration and invasion assays showing that knockdown of GRB7 inhibited cell proliferation, migration and invasion. (D) The 3-4-week-old female nude mice were i.p . injected with GRB7 knockdown or scrambled control SKOV3 cells and photographed on Day 60. Yellow arrows indicate the location of tumor nodules. The number of tumor nodules on the last day in each group are illustrated by bar charts (N=5).

Article Snippet: Ovarian cancer cells with stable GRB7 knockdown were infected with lentiviral particles carrying GRB7 shRNA plasmid (Santa Cruz).

Techniques: Functional Assay, Knockdown, Migration, In Vivo, Western Blot, Control, Proliferation Assay, Injection

Journal: Theranostics

Article Title: Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness by targeting GRB7 and MAPK/ERK pathways

doi: 10.7150/thno.22377

Figure Lengend Snippet: DOX-induced miR-193a-3p inhibits cell migration and invasion in vitro and results in complete inhibition of tumor growth. (A) qPCR and (B) Western blot analysis showing induction of the expression of GRB7 by 1 μg/ml doxycycline (Dox) in a time-dependent manner. SNORD48 was used as an internal control. (C) XTT cell proliferation assay showing the effect of inducible miR-193a-3p on SKOV3 cells. (D) Transwell cell migration/invasion assays demonstrating the suppressive effect of inducible miR-193a-3p on the migration and invasion capacities of SKOV3 ovarian cancer cells. The induction of miR-193a-3p for cell proliferation and cell migration/invasion using 1 μg/ml Dox. (E) Schematic overview of miR-193a-3p expression in mice and its effect on the suppression of ovarian tumor growth in the intraperitoneal cavity of nude mice. (F) The 3-4-week-old female mice received an intraperitoneal injection (i.p.) of 1x10 6 miR-193a-3p -inducible SKOV3 cells. When palpable tumors formed on Day 20, all mice were treated with or without 2 mg/mL Dox in the drinking water until they were sacrificed on Day 60. Representative photographs of tumor nodules collected after induction of miR-193a-3p on Day 60. The table shows that only 1 out of 4 mice had tumor nodules in the Dox-inducible miR-193a-3p group, while all mice (4 out of 4) developed tumor nodules in the control group. The number of tumor nodules on the last day is illustrated for each group by bar charts (N=4).

Article Snippet: Ovarian cancer cells with stable GRB7 knockdown were infected with lentiviral particles carrying GRB7 shRNA plasmid (Santa Cruz).

Techniques: Migration, In Vitro, Inhibition, Western Blot, Expressing, Control, Proliferation Assay, Injection

Journal: PLoS ONE

Article Title: Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

doi: 10.1371/journal.pone.0052578

Figure Lengend Snippet: Clinico-pathological analysis of GRB7, ERK phosphorylation and FOXM1 expressions in ovarian cancer tissue array (OVC1021).

Article Snippet: Four shRNA HuSH 29mer shRNA constructs against GRB7 in pGFP-V-RS vector were purchased from OriGene Technologies for generating stable GRB7 knockdown cells (Cat. No. TG312621, OriGene Technologies, Inc, Rockville, MD, USA).

Techniques: Phospho-proteomics

Journal: PLoS ONE

Article Title: Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

doi: 10.1371/journal.pone.0052578

Figure Lengend Snippet: (A) The expressions of GRB7, ERK phosphorylation and FOXM1 were evaluated by immunohistochemical analysis using specific antibodies on an ovarian cancer tissue array (OVC1021, Pantomics). (B) Representative pictures show the stepwise increase of GRB7, ERK phosphorylation and FOXM1 expressions from Grade 1 to Grade 3 ovarian cancer (serous subtype) (200× magnifications).

Article Snippet: Four shRNA HuSH 29mer shRNA constructs against GRB7 in pGFP-V-RS vector were purchased from OriGene Technologies for generating stable GRB7 knockdown cells (Cat. No. TG312621, OriGene Technologies, Inc, Rockville, MD, USA).

Techniques: Phospho-proteomics, Immunohistochemical staining

Journal: PLoS ONE

Article Title: Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

doi: 10.1371/journal.pone.0052578

Figure Lengend Snippet: (A) Treatment with U0126 (5 µM) showed a significant reduction in the expression of ERK phosphorylation accompanied with FOXM1 in ovarian cancer cells time dependently, whereas no change in GRB7 expression was found in A2780cp cells. (B) Treatment of Thiostrepton (20 µM) remarkably reduced the expression of FOXM1 only but no change in the expressions of GRB7 and ERK phosphorylation in A2780cp cells ( left ). Depletion of FOXM1 by siRNA knockdown did not alter the expression of GRB7 and ERK phosphorylation ( right ). C, siRNA scrambled control. si1, si2 and si3 siRNAs targeting three different regions of human FOXM1 and knockdown the expression of FOXM1 by 60%, 45% and 70% respectively. (C) Two out of four GRB7 shRNA constructs (sh1 and sh2) showed ∼70% knockdown of GRB7 accompanied with a reduction of ERK phosphorylation and FOXM1 expressions in OVCA433 cells. The scrambled control (NC) was used as negative control. (D) Enforced expression of GRB7 increased ERK phosphorylation and FOXM1. However, treatment with either U0126 (10 µM) or PD98059 (20 µM) could suppress the induced ERK phosphorylation and FOXM1 in A2780cp and OVCA433 ovarian cancer cells.

Article Snippet: Four shRNA HuSH 29mer shRNA constructs against GRB7 in pGFP-V-RS vector were purchased from OriGene Technologies for generating stable GRB7 knockdown cells (Cat. No. TG312621, OriGene Technologies, Inc, Rockville, MD, USA).

Techniques: Expressing, Phospho-proteomics, Knockdown, Control, shRNA, Construct, Negative Control

Journal: PLoS ONE

Article Title: Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

doi: 10.1371/journal.pone.0052578

Figure Lengend Snippet: OVCA433 cells with stable expression of GFP/GRB7 (OVCA433-GRB7) were treated with DMSO as control, Thiostrepton (20 µM), PD98059 (20 µM) and U0126 (10 µM) for 6 hours and were analyzed by (A) Transwell cell migration assay. The representative pictures and bar chart showed significant reduction in the number of migratory cells through Matrigel-coated membrane in OVCA433-GRB7 cells treated with Thiostrepton, PD98059 and U0126 than DMSO control (* P <0.02, Student t -test) at 8-hour; (B) Transwell cell invasion assay. The representative pictures and bar chart showed significant reduction in the invasion rate in OVCA433-GRB7 cells treated with Thiostrepton, PD98059 and U0126 when compared with DMSO control (* P <0.05, Student t -test) at 15-hour.

Article Snippet: Four shRNA HuSH 29mer shRNA constructs against GRB7 in pGFP-V-RS vector were purchased from OriGene Technologies for generating stable GRB7 knockdown cells (Cat. No. TG312621, OriGene Technologies, Inc, Rockville, MD, USA).

Techniques: Expressing, Control, Cell Migration Assay, Membrane, Invasion Assay

Journal: PLoS ONE

Article Title: Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

doi: 10.1371/journal.pone.0052578

Figure Lengend Snippet: (A) XTT cell proliferation assays showed that the inhibition of ERK phosphorylation by U0126 (10 µM) significantly abrogated the cell proliferation rate in GRB7 stably expressing OVCA433 cells ( P = 0.020, Student t -test) and A2780cp cells ( P = 0.030, Student t -test) as compared with their vector controls. (B) XTT cell proliferation assays showed that the suppression of FOXM1 expression by Thiostrepton (20 µM) significantly reduced the cell proliferation rate in GRB7 stably expressing OVCA433 cells ( P = 0.015, Student t -test) and A2780cp cells ( P = 0.025, Student t -test) as compared with their vector controls.

Article Snippet: Four shRNA HuSH 29mer shRNA constructs against GRB7 in pGFP-V-RS vector were purchased from OriGene Technologies for generating stable GRB7 knockdown cells (Cat. No. TG312621, OriGene Technologies, Inc, Rockville, MD, USA).

Techniques: Inhibition, Phospho-proteomics, Stable Transfection, Expressing, Plasmid Preparation

Journal: PLoS ONE

Article Title: Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

doi: 10.1371/journal.pone.0052578

Figure Lengend Snippet: (A) The GRB7 stably expressing A2780cp cells (Acp-GRB7) were subcutaneously injected into the right flank of nude mice. Mice were divided into 5 groups (5 mice per group) and treated with either DMSO as a control, or U0126 (25 or 50 µM/kg) or Thiostrepton (200 or 300 µM/kg) for every 3-day since on day 6 (Arrows represent the injections). The relative tumor size was calculated relative to those of the first day of treatment (day 0) and are represented as relative mean size (%)±SE for each group (* P = 0. 032, ** P <0.01, and *** P = 0. 005, are significantly different from the DMSO control group, Student t -test). (B) The representative pictures and bar charts show the average tumor weight of each group taken on day 18. (* P = 0. 043, ** P = 0. 001, and *** P <0.02, are significantly different from the DMSO control group, Student t -test).

Article Snippet: Four shRNA HuSH 29mer shRNA constructs against GRB7 in pGFP-V-RS vector were purchased from OriGene Technologies for generating stable GRB7 knockdown cells (Cat. No. TG312621, OriGene Technologies, Inc, Rockville, MD, USA).

Techniques: Stable Transfection, Expressing, Injection, Control

Journal: Oncotarget

Article Title: The prognostic significance of GRB7 protein expression and localization in human breast and ovarian cancers.

doi: 10.18632/oncotarget.27593

Figure Lengend Snippet: Figure 1: GRB7 expression in normal breast and ovarian tissues. Representative images of normal tissues stained with the GRB7 antibody (brown) and Hematoxylin (blue). (A) Normal breast tissue showing no GRB7 expression in the mammary ducts. (B) Normal breast tissue showing high GRB7 expression (3+) in the myoepithelial cells of mammary ducts. (C) Normal ovarian surface epithelium showing strong cytoplasmic GRB7 (3+) expression. (D) Normal ovarian tissue showing no GRB7 expression in the stroma. (E) Normal fallopian tubes with weak GRB7 (1+) expression. All images were taken with a 40× objective (scale bar 50 µm).

Article Snippet: anti-human GRB7, rabbit monoclonal antibody (Abcam, Cat# 183737 [EPR14099]); anti α-tubulin, mouse Oncotarget2287www.oncotarget.com monoclonal antibody (Cell signaling, Danvers, MA, USA, Cat# 3873s); anti HER-2/ErbB2, rabbit polyclonal antibody (Cell signaling, Cat# 2242).

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: The prognostic significance of GRB7 protein expression and localization in human breast and ovarian cancers.

doi: 10.18632/oncotarget.27593

Figure Lengend Snippet: Figure 2: GRB7 expression in benign fibroadenoma and fibrocystic changes. Examples of images of benign breast cancer tumors stained with the GRB7 antibody (brown) and Hematoxylin (blue). (A) Benign fibroadenoma tissue showing high GRB7 expression (3+) in myoepithelial cells. (B) Benign fibrocystic changes showing no GRB7 expression (0). Images were taken with a 40 × objective, scale bar = 50 µm).

Article Snippet: anti-human GRB7, rabbit monoclonal antibody (Abcam, Cat# 183737 [EPR14099]); anti α-tubulin, mouse Oncotarget2287www.oncotarget.com monoclonal antibody (Cell signaling, Danvers, MA, USA, Cat# 3873s); anti HER-2/ErbB2, rabbit polyclonal antibody (Cell signaling, Cat# 2242).

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: The prognostic significance of GRB7 protein expression and localization in human breast and ovarian cancers.

doi: 10.18632/oncotarget.27593

Figure Lengend Snippet: Figure 3: GRB7 expression and localization in invasive ductal carcinoma (IDC) of the breast. Representative images of IDC of the breast tissues stained with the GRB7 antibody (brown) and Hematoxylin (blue). (A) IDC showing no GRB7 expression (0). (B) IDC showing weak cytoplasmic GRB7 expression (1+). (C) IDC showing medium cytoplasmic GRB7 expression (2+). (D) IDC showing strong cytoplasmic GRB7 expression (3+). (E) IDC showing strong cytoplasmic GRB7 expression with membranous accentuation (3+). Images were taken with a 40 × objective, scale bar = 50 µm.

Article Snippet: anti-human GRB7, rabbit monoclonal antibody (Abcam, Cat# 183737 [EPR14099]); anti α-tubulin, mouse Oncotarget2287www.oncotarget.com monoclonal antibody (Cell signaling, Danvers, MA, USA, Cat# 3873s); anti HER-2/ErbB2, rabbit polyclonal antibody (Cell signaling, Cat# 2242).

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: The prognostic significance of GRB7 protein expression and localization in human breast and ovarian cancers.

doi: 10.18632/oncotarget.27593

Figure Lengend Snippet: Figure 4: GRB7 expression in ovarian carcinoma cells. Representative images of high grade ovarian cancer tissues stained with the GRB7 antibody (brown) and Hematoxylin (blue). (A) No GRB7 expression (0). (B) Weak cytoplasmic GRB7 expression (1+). (C) Medium cytoplasmic GRB7 expression (2+). (D) Strong cytoplasmic GRB7 expression (3+). (E) Strong cytoplasmic GRB7 expression with membranous accentuation (3+). Images were taken with a 40 × objective, scale bar = 50 µm.

Article Snippet: anti-human GRB7, rabbit monoclonal antibody (Abcam, Cat# 183737 [EPR14099]); anti α-tubulin, mouse Oncotarget2287www.oncotarget.com monoclonal antibody (Cell signaling, Danvers, MA, USA, Cat# 3873s); anti HER-2/ErbB2, rabbit polyclonal antibody (Cell signaling, Cat# 2242).

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: The prognostic significance of GRB7 protein expression and localization in human breast and ovarian cancers.

doi: 10.18632/oncotarget.27593

Figure Lengend Snippet: Figure 5: Kaplan-Meier survival analysis for GRB7 membrane-associated staining from 173 patients with ovarian cancer. (A) Recurrence Free Survival (RFS) and (B) Overall Survival (OS) curves for each patient showing GRB7 membrane-associated expression shown as yes (black curve)/no (gray curve) for each patient). (C) Landmark analysis for RFS (shown in (A) using 810 days (27 months) as the cut off mark (dotted lined). (D) RFS and (E) OS curves for patients with at least two out of three tissue cores with GRB7 membrane-associated expression (black curve) vs. one or no GRB7 membrane-associated expression (gray curve). All survival curves were generated after dichotomization of GRB7 membrane-associated expression (black curves, “GRB7 yes”) or no GRB7 membrane-associated expression (gray curve, “GRB7 no”), obtained by IHC. p-values were calculated by log-rank tests.

Article Snippet: anti-human GRB7, rabbit monoclonal antibody (Abcam, Cat# 183737 [EPR14099]); anti α-tubulin, mouse Oncotarget2287www.oncotarget.com monoclonal antibody (Cell signaling, Danvers, MA, USA, Cat# 3873s); anti HER-2/ErbB2, rabbit polyclonal antibody (Cell signaling, Cat# 2242).

Techniques: Membrane, Staining, Expressing, Generated, Paraffin-embedded Immunohistochemistry

Journal: Oncotarget

Article Title: The prognostic significance of GRB7 protein expression and localization in human breast and ovarian cancers.

doi: 10.18632/oncotarget.27593

Figure Lengend Snippet: Figure 6: GRB7 expression levels and localization of breast cancer cell lines (MDA-MB-231, HCC1954, and SKBR3). (A) Western blot showing GRB7 expression levels (middle) panel using total protein lysates (10 µg/lane). Alpha-tubulin (aTUB, bottom panel) was used as internal reference, HER-2 was used to show co-amplification of HER-2 and GRB7 as expected (top panel). MD = MDA-MB-231, HC = HCC1954, SK = SKBR3. (B) Representative images of breast cancer (HCC1954, SKBR3) cell lines stained (IHC) with the GRB7 antibody (brown, developed using HRP-DAB) and nuclei (Hematoxylin, blue). Images show cells with strong cytoplasmic GRB7 expression with membranous accentuation. (C) Representative images of breast cancer (HCC1954, SKBR3) cell lines stained (IF) with the GRB7 antibody (green), WGA cell membrane marker (magenta), nuclei (blue), and a merged image from all three channels, showing GRB7 and WGA co-localization in white (most right). A DIC image is shown on the most left. Images were taken with a 20 × objective, scale bar = 100 µm.

Article Snippet: anti-human GRB7, rabbit monoclonal antibody (Abcam, Cat# 183737 [EPR14099]); anti α-tubulin, mouse Oncotarget2287www.oncotarget.com monoclonal antibody (Cell signaling, Danvers, MA, USA, Cat# 3873s); anti HER-2/ErbB2, rabbit polyclonal antibody (Cell signaling, Cat# 2242).

Techniques: Expressing, Western Blot, Amplification, Staining, Membrane, Marker

Journal: PloS one

Article Title: Targeting GRB7/ERK/FOXM1 signaling pathway impairs aggressiveness of ovarian cancer cells.

doi: 10.1371/journal.pone.0052578

Figure Lengend Snippet: Figure 1. A significant stepwise increase in the expressions of GRB7, ERK phosphorylation and FOXM1 along ovarian tumor grade. (A) The expressions of GRB7, ERK phosphorylation and FOXM1 were evaluated by immunohistochemical analysis using specific antibodies on an ovarian cancer tissue array (OVC1021, Pantomics). (B) Representative pictures show the stepwise increase of GRB7, ERK phosphorylation and FOXM1 expressions from Grade 1 to Grade 3 ovarian cancer (serous subtype) (2006 magnifications). doi:10.1371/journal.pone.0052578.g001

Article Snippet: Four shRNA HuSH 29mer shRNA constructs against GRB7 in pGFP-V-RS vector were purchased from OriGene Technologies for generating stable GRB7 knockdown cells (Cat. No. TG312621, OriGene Technologies, Inc, Rockville, MD, USA).

Techniques: Phospho-proteomics, Immunohistochemical staining

Journal: PloS one

Article Title: Targeting GRB7/ERK/FOXM1 signaling pathway impairs aggressiveness of ovarian cancer cells.

doi: 10.1371/journal.pone.0052578

Figure Lengend Snippet: Figure 2. GRB7/ERK/FOXM1 was regulated in the same signaling axis. (A) Treatment with U0126 (5 mM) showed a significant reduction in the expression of ERK phosphorylation accompanied with FOXM1 in ovarian cancer cells time dependently, whereas no change in GRB7 expression was found in A2780cp cells. (B) Treatment of Thiostrepton (20 mM) remarkably reduced the expression of FOXM1 only but no change in the expressions of GRB7 and ERK phosphorylation in A2780cp cells (left). Depletion of FOXM1 by siRNA knockdown did not alter the expression of GRB7 and ERK phosphorylation (right). C, siRNA scrambled control. si1, si2 and si3 siRNAs targeting three different regions of human FOXM1 and knockdown the expression of FOXM1 by 60%, 45% and 70% respectively. (C) Two out of four GRB7 shRNA constructs (sh1 and sh2) showed ,70% knockdown of GRB7 accompanied with a reduction of ERK phosphorylation and FOXM1 expressions in OVCA433 cells. The scrambled control (NC) was used as negative control. (D) Enforced expression of GRB7 increased ERK phosphorylation and FOXM1. However, treatment with either U0126 (10 mM) or PD98059 (20 mM) could suppress the induced ERK phosphorylation and FOXM1 in A2780cp and OVCA433 ovarian cancer cells. doi:10.1371/journal.pone.0052578.g002

Article Snippet: Four shRNA HuSH 29mer shRNA constructs against GRB7 in pGFP-V-RS vector were purchased from OriGene Technologies for generating stable GRB7 knockdown cells (Cat. No. TG312621, OriGene Technologies, Inc, Rockville, MD, USA).

Techniques: Expressing, Phospho-proteomics, Knockdown, Control, shRNA, Construct, Negative Control

Journal: PloS one

Article Title: Targeting GRB7/ERK/FOXM1 signaling pathway impairs aggressiveness of ovarian cancer cells.

doi: 10.1371/journal.pone.0052578

Figure Lengend Snippet: Figure 3. Inhibition of ERK phosphorylation or FOXM1 significantly decreased both migration and invasion of GRB7- overexpressing ovarian cancer cells. OVCA433 cells with stable expression of GFP/GRB7 (OVCA433-GRB7) were treated with DMSO as control, Thiostrepton (20 mM), PD98059 (20 mM) and U0126 (10 mM) for 6 hours and were analyzed by (A) Transwell cell migration assay. The representative pictures and bar chart showed significant reduction in the number of migratory cells through Matrigel-coated membrane in OVCA433-GRB7 cells treated with Thiostrepton, PD98059 and U0126 than DMSO control (*P,0.02, Student t-test) at 8-hour; (B) Transwell cell invasion assay. The representative pictures and bar chart showed significant reduction in the invasion rate in OVCA433-GRB7 cells treated with Thiostrepton, PD98059 and U0126 when compared with DMSO control (*P,0.05, Student t-test) at 15-hour. doi:10.1371/journal.pone.0052578.g003

Article Snippet: Four shRNA HuSH 29mer shRNA constructs against GRB7 in pGFP-V-RS vector were purchased from OriGene Technologies for generating stable GRB7 knockdown cells (Cat. No. TG312621, OriGene Technologies, Inc, Rockville, MD, USA).

Techniques: Inhibition, Phospho-proteomics, Migration, Expressing, Control, Cell Migration Assay, Membrane, Invasion Assay

Journal: PloS one

Article Title: Targeting GRB7/ERK/FOXM1 signaling pathway impairs aggressiveness of ovarian cancer cells.

doi: 10.1371/journal.pone.0052578

Figure Lengend Snippet: Figure 4. Inhibition of either ERK phosphorylation or FOXM1 expression impaired cell growth in ovarian cancer cells. (A) XTT cell proliferation assays showed that the inhibition of ERK phosphorylation by U0126 (10 mM) significantly abrogated the cell proliferation rate in GRB7 stably expressing OVCA433 cells (P = 0.020, Student t-test) and A2780cp cells (P = 0.030, Student t-test) as compared with their vector controls. (B) XTT cell proliferation assays showed that the suppression of FOXM1 expression by Thiostrepton (20 mM) significantly reduced the cell proliferation rate in GRB7 stably expressing OVCA433 cells (P = 0.015, Student t-test) and A2780cp cells (P = 0.025, Student t-test) as compared with their vector controls. doi:10.1371/journal.pone.0052578.g004

Article Snippet: Four shRNA HuSH 29mer shRNA constructs against GRB7 in pGFP-V-RS vector were purchased from OriGene Technologies for generating stable GRB7 knockdown cells (Cat. No. TG312621, OriGene Technologies, Inc, Rockville, MD, USA).

Techniques: Inhibition, Phospho-proteomics, Expressing, Stable Transfection, Plasmid Preparation

Journal: PloS one

Article Title: Targeting GRB7/ERK/FOXM1 signaling pathway impairs aggressiveness of ovarian cancer cells.

doi: 10.1371/journal.pone.0052578

Figure Lengend Snippet: Figure 5. Inhibition of ERK phosphorylation or FOXM1 expression reduced tumor growth in a mouse xenograft model. (A) The GRB7 stably expressing A2780cp cells (Acp-GRB7) were subcutaneously injected into the right flank of nude mice. Mice were divided into 5 groups (5 mice per group) and treated with either DMSO as a control, or U0126 (25 or 50 mM/kg) or Thiostrepton (200 or 300 mM/kg) for every 3-day since on day 6

Article Snippet: Four shRNA HuSH 29mer shRNA constructs against GRB7 in pGFP-V-RS vector were purchased from OriGene Technologies for generating stable GRB7 knockdown cells (Cat. No. TG312621, OriGene Technologies, Inc, Rockville, MD, USA).

Techniques: Inhibition, Phospho-proteomics, Expressing, Stable Transfection, Injection, Control