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  • 94
    Thermo Fisher egfr
    Egfr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore egfr
    Egfr, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1033 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc egfr
    Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5919 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti egfr
    Effects of oridonin on the binding probability for <t>EGF-EGFR</t> complex in KYSE-150 cells, data was expressed as mean ± S.E.M. from five independent experiments, and in each experiments, more than 1000 force curves were calculated, *p
    Anti Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1758 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology egfr
    Overexpression of <t>TIEG1</t> attenuates <t>EGFR</t> expression and histone acetylation of the EGFR promoter in MDA-MB-231HM cells. (A and B) TIEG1 expression vector or control vector was transfected into MDA-MB-231HM cells and generated stable transfectants, MDA-MB-231HM/TIEG1
    Egfr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 3488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc p egfr
    RHOB induces resistance to erlotinib through the AKT pathway HCC4006, HCC827, HCC2935, and H3255 cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 4 h with erlotinib at concentrations corresponding to the respective IC 50 values determined for each control cell line. The phosphorylation status of AKT, <t>ERK1/2,</t> and <t>EGFR</t> was assessed by Western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by Western blotting. Representative immunostaining of phospho‐AKT (Ser473) and phospho‐ERK1/2 and their total protein amounts in lung tumors from EGFR L858R / Rhob − / − or EGFR L858R / Rhob +/+ mice treated or not with erlotinib (12.5 mg/kg/day) for 4 days. The remaining hyperplastic areas were selected in erlotinib‐treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bars: 100 μm. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKT myr , myristoylated) or an empty vector (ø) and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay, and AKT overexpression and phosphorylation at Ser473 were assessed by Western blotting. Data are representative of at least three independent experiments. Data are expressed as mean ± SEM from three independent experiments. Source data are available online for this figure.
    P Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p egfr/product/Cell Signaling Technology Inc
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    egfr  (Abcam)
    99
    Abcam egfr
    RHOB induces resistance to erlotinib through the AKT pathway HCC4006, HCC827, HCC2935, and H3255 cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 4 h with erlotinib at concentrations corresponding to the respective IC 50 values determined for each control cell line. The phosphorylation status of AKT, <t>ERK1/2,</t> and <t>EGFR</t> was assessed by Western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by Western blotting. Representative immunostaining of phospho‐AKT (Ser473) and phospho‐ERK1/2 and their total protein amounts in lung tumors from EGFR L858R / Rhob − / − or EGFR L858R / Rhob +/+ mice treated or not with erlotinib (12.5 mg/kg/day) for 4 days. The remaining hyperplastic areas were selected in erlotinib‐treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bars: 100 μm. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKT myr , myristoylated) or an empty vector (ø) and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay, and AKT overexpression and phosphorylation at Ser473 were assessed by Western blotting. Data are representative of at least three independent experiments. Data are expressed as mean ± SEM from three independent experiments. Source data are available online for this figure.
    Egfr, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 900 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc total egfr
    RHOB induces resistance to erlotinib through the AKT pathway HCC4006, HCC827, HCC2935, and H3255 cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 4 h with erlotinib at concentrations corresponding to the respective IC 50 values determined for each control cell line. The phosphorylation status of AKT, <t>ERK1/2,</t> and <t>EGFR</t> was assessed by Western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by Western blotting. Representative immunostaining of phospho‐AKT (Ser473) and phospho‐ERK1/2 and their total protein amounts in lung tumors from EGFR L858R / Rhob − / − or EGFR L858R / Rhob +/+ mice treated or not with erlotinib (12.5 mg/kg/day) for 4 days. The remaining hyperplastic areas were selected in erlotinib‐treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bars: 100 μm. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKT myr , myristoylated) or an empty vector (ø) and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay, and AKT overexpression and phosphorylation at Ser473 were assessed by Western blotting. Data are representative of at least three independent experiments. Data are expressed as mean ± SEM from three independent experiments. Source data are available online for this figure.
    Total Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 444 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies egfr
    Kaplan Meier curves of disease free survival of risk groups for TNBC patients. The patients were stratified into different risk groups. Two low basal <t>(EGFR≤15%</t> and <t>CK5/6≤50%)</t> risk groups: group 1 (n = 20) with low expressions/values of EGFR (≤15%), CK5/6 (≤50%), Ki-67 (≤50%), pT (≤ 3), and pN (≤ 1), and group 2 (n = 30) with low expressions of EGFR and CK5/6, and any high expressions/values of Ki-67, pT, and pN. Two high basal risk groups: group 3 (n = 106) with single high basal expression (EGFR > 15% or CK5/6 > 50%), and group 4 (n = 36) with double high basal expressions (EGFR > 15% and CK5/6 > 50%).
    Egfr, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 1139 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen therascreen egfr rgq pcr kit
    Kaplan Meier curves of disease free survival of risk groups for TNBC patients. The patients were stratified into different risk groups. Two low basal <t>(EGFR≤15%</t> and <t>CK5/6≤50%)</t> risk groups: group 1 (n = 20) with low expressions/values of EGFR (≤15%), CK5/6 (≤50%), Ki-67 (≤50%), pT (≤ 3), and pN (≤ 1), and group 2 (n = 30) with low expressions of EGFR and CK5/6, and any high expressions/values of Ki-67, pT, and pN. Two high basal risk groups: group 3 (n = 106) with single high basal expression (EGFR > 15% or CK5/6 > 50%), and group 4 (n = 36) with double high basal expressions (EGFR > 15% and CK5/6 > 50%).
    Therascreen Egfr Rgq Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 588 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Abcam anti egfr
    Kaplan Meier curves of disease free survival of risk groups for TNBC patients. The patients were stratified into different risk groups. Two low basal <t>(EGFR≤15%</t> and <t>CK5/6≤50%)</t> risk groups: group 1 (n = 20) with low expressions/values of EGFR (≤15%), CK5/6 (≤50%), Ki-67 (≤50%), pT (≤ 3), and pN (≤ 1), and group 2 (n = 30) with low expressions of EGFR and CK5/6, and any high expressions/values of Ki-67, pT, and pN. Two high basal risk groups: group 3 (n = 106) with single high basal expression (EGFR > 15% or CK5/6 > 50%), and group 4 (n = 36) with double high basal expressions (EGFR > 15% and CK5/6 > 50%).
    Anti Egfr, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho egfr tyr1068
    Kaplan Meier curves of disease free survival of risk groups for TNBC patients. The patients were stratified into different risk groups. Two low basal <t>(EGFR≤15%</t> and <t>CK5/6≤50%)</t> risk groups: group 1 (n = 20) with low expressions/values of EGFR (≤15%), CK5/6 (≤50%), Ki-67 (≤50%), pT (≤ 3), and pN (≤ 1), and group 2 (n = 30) with low expressions of EGFR and CK5/6, and any high expressions/values of Ki-67, pT, and pN. Two high basal risk groups: group 3 (n = 106) with single high basal expression (EGFR > 15% or CK5/6 > 50%), and group 4 (n = 36) with double high basal expressions (EGFR > 15% and CK5/6 > 50%).
    Phospho Egfr Tyr1068, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology p egfr
    LMP1 increased the binding ability of transcription factors <t>EGFR</t> and <t>STAT3</t> to cyclin D1 promoter in vitro . (A) STAT3 binding activities within the cyclin D1 promoter were examined by EMSA. A biotin-labeled wild-type STAT3 oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type STAT3 (lane 4), unlabeled mutant STAT3 oligonucleotides (lane 5), or noncompetitive unlabeled NF-κB oligonucleotide (NS, lane 6). Biotin-labeled mutant STAT3 oligonucleotide probe was incubated with nuclear extracts of the indicated NPC cell lines (lanes 8–9). (B) Ten micrograms of nuclear extracts were pre-incubated with biotin-labeled STAT3 oligonucleotide probe in the presence of inhibitors directed against different phosphorylation sites of STAT3 (indicated above each lane). (C) The biotin-labeled wild-type EGFR oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type EGFR (lane 4), unlabeled mutant EGFR oligonucleotides (lane 6) or noncompetitive unlabeled NFκB oligonucleotide (NS, lane 7), and then EGFR DNA binding activities were examined by EMSA. (D-E) The nuclear extracts of CNE1 and CNE1-LMP1 cells were pre-incubated with biotin-labeled EGFR oligonucleotide probe in the presence of inhibitors AG1478, directed against phosphorylation of EGFR, or DNAzyme 1 (DZ1), targeting LMP1. RD: relative density.
    P Egfr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 379 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti egfr
    Resistance to Th1 <t>cytokine/trastuzumab-mediated</t> class I restoration and HER2 369-377 -CD8 + T-cell targeting of HER2-expressing cancers by EGF/Heregulin is rescued with inhibition of <t>EGFR</t> and HER3 signaling (A) Effect of EGFR- and HER3-mediated signaling on class I restoration by trastuzumab and Th1 cytokines. Trastuzumab-treated HER2 high BT-474 cells were serum starved and activated with EGF, Heregulin, or both, followed by IFNγ and TNFα treatment. Harvested cells were assessed for HLA-ABC expression by flow cytometry. Results are representative of three experiments, and expressed as mean HLA-ABC MCF ± SEM. (B) Trastuzumab/IFNγ/TNFα-treated HER2 high SK-BR-3 ( left panel ) and BT-474 ( right panel ) cells with or without EGF + Heregulin activation, were subsequently treated with anti-EGFR + anti-HER3 neutralizing or IgG1 isotype control antibodies. Harvested cells were assessed for HLA-ABC expression by flow cytometry. In representative panels, filled traces represent isotype-matched control staining, and open traces represent HLA-ABC staining. Color-coded cell treatments are indicated in the legend. Adjoining results in histograms are representative of three experiments, and expressed as mean HLA-ABC MCF ± SEM; cell treatments are indicated below the histograms. (C) Following treatments indicated in (B) above, CFSE-labeled HER2 high SK-BR-3 cells were co-cultured 1:1 with HER2 369-377 -sensitized CD8 + T cells. Tumor cells were harvested, stained with 7-AAD and FITC:anti-CD8, and CSFE + 7-AAD + CD8 - cells (apoptotic) were assessed by flow cytometry. Results are representative of three experiments, and expressed as % apoptotic tumor cells±SEM ( % lysis ) in co-culture minus background. *p≤0.05, **p
    Anti Egfr, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti egfr
    Downregulation of <t>EGFR</t> and <t>p1068EGFR</t> expression by EGCG and IIF treatments in BE(2)-C neuroblastoma cells. Cells were treated with 20 μg/mL EGCG and 10 μM IIF, individually and in combination for 24 h. ( A ) Proteins (50 μg) from total cell lysate were subjected to SDS–PAGE and Western blot analysis of EGFR and p1068EGFR expression after 24 h treatments. Actin was used as a loading control. RT-PCR analysis of EGFR ( B ) and NDRG1 ( C ) in control and treated cells. β-actin was used as a control. The values were normalized to the untreated controls. The results are expressed as the average ± SE of three independent experiments. * p
    Anti Egfr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of oridonin on the binding probability for EGF-EGFR complex in KYSE-150 cells, data was expressed as mean ± S.E.M. from five independent experiments, and in each experiments, more than 1000 force curves were calculated, *p

    Journal: Pharmacological research

    Article Title: Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells

    doi: 10.1016/j.phrs.2016.11.036

    Figure Lengend Snippet: Effects of oridonin on the binding probability for EGF-EGFR complex in KYSE-150 cells, data was expressed as mean ± S.E.M. from five independent experiments, and in each experiments, more than 1000 force curves were calculated, *p

    Article Snippet: EGF was purchased from R & D (USA) and anti-EGFR antibody was obtained from Cell Signaling (USA).

    Techniques: Binding Assay

    Unbinding forces measured on the surface of KYSE-150 cells by EGF-functionalized AFM tips. Histogram of unbinding forces of EGF-EGFR complexes obtained on (A) control KYSE-150 cells, (B) 10 μM oridonin treated KYSE-150 cells, (C) 30 μM

    Journal: Pharmacological research

    Article Title: Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells

    doi: 10.1016/j.phrs.2016.11.036

    Figure Lengend Snippet: Unbinding forces measured on the surface of KYSE-150 cells by EGF-functionalized AFM tips. Histogram of unbinding forces of EGF-EGFR complexes obtained on (A) control KYSE-150 cells, (B) 10 μM oridonin treated KYSE-150 cells, (C) 30 μM

    Article Snippet: EGF was purchased from R & D (USA) and anti-EGFR antibody was obtained from Cell Signaling (USA).

    Techniques:

    Effects of oridonin on the dynamic force spectra for EGF-EGFR complex in KYSE-150 cells. (A) Control KYSE-150 cells, (B) 10 μM oridonin treated KYSE-150 cells,(C) 30 μM oridonin treated KYSE-150 cells, (D) 50 μM oridonin treated

    Journal: Pharmacological research

    Article Title: Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells

    doi: 10.1016/j.phrs.2016.11.036

    Figure Lengend Snippet: Effects of oridonin on the dynamic force spectra for EGF-EGFR complex in KYSE-150 cells. (A) Control KYSE-150 cells, (B) 10 μM oridonin treated KYSE-150 cells,(C) 30 μM oridonin treated KYSE-150 cells, (D) 50 μM oridonin treated

    Article Snippet: EGF was purchased from R & D (USA) and anti-EGFR antibody was obtained from Cell Signaling (USA).

    Techniques:

    Schematic diagram illustrating the molecular mechanism of oridonin inhibited EGF-EGFR binding and cell death by ROS dependent way in KYSE-150 cells. F, P, x β , k off and ΔG is the unbinding force, binding probability, energy barrier width,

    Journal: Pharmacological research

    Article Title: Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells

    doi: 10.1016/j.phrs.2016.11.036

    Figure Lengend Snippet: Schematic diagram illustrating the molecular mechanism of oridonin inhibited EGF-EGFR binding and cell death by ROS dependent way in KYSE-150 cells. F, P, x β , k off and ΔG is the unbinding force, binding probability, energy barrier width,

    Article Snippet: EGF was purchased from R & D (USA) and anti-EGFR antibody was obtained from Cell Signaling (USA).

    Techniques: Binding Assay

    AFM force measurements with EGF-functionalized AFM tip on living KYSE-150 cells. (A) AFM tip functionalization procedure and schematic representation of the single-molecule force measurement between EGF-functionalized AFM tips and EGFR in KYSE-150 cells.

    Journal: Pharmacological research

    Article Title: Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells

    doi: 10.1016/j.phrs.2016.11.036

    Figure Lengend Snippet: AFM force measurements with EGF-functionalized AFM tip on living KYSE-150 cells. (A) AFM tip functionalization procedure and schematic representation of the single-molecule force measurement between EGF-functionalized AFM tips and EGFR in KYSE-150 cells.

    Article Snippet: EGF was purchased from R & D (USA) and anti-EGFR antibody was obtained from Cell Signaling (USA).

    Techniques:

    Histogram distribution of the rupture length for EGF-EGFR complex in KYSE-150 cells. Rupture length for EGF-EGFR complex obtained on (A) control KYSE-150 cells, (B) 10 μM oridonin treated KYSE-150 cells, (C) 30 μM oridonin treated KYSE-150

    Journal: Pharmacological research

    Article Title: Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells

    doi: 10.1016/j.phrs.2016.11.036

    Figure Lengend Snippet: Histogram distribution of the rupture length for EGF-EGFR complex in KYSE-150 cells. Rupture length for EGF-EGFR complex obtained on (A) control KYSE-150 cells, (B) 10 μM oridonin treated KYSE-150 cells, (C) 30 μM oridonin treated KYSE-150

    Article Snippet: EGF was purchased from R & D (USA) and anti-EGFR antibody was obtained from Cell Signaling (USA).

    Techniques:

    3.4. Effects of oridonin on the dynamic force spectra and dissociation kinetics for EGF-EGFR complex in KYSE-150 cells

    Journal: Pharmacological research

    Article Title: Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells

    doi: 10.1016/j.phrs.2016.11.036

    Figure Lengend Snippet: 3.4. Effects of oridonin on the dynamic force spectra and dissociation kinetics for EGF-EGFR complex in KYSE-150 cells

    Article Snippet: EGF was purchased from R & D (USA) and anti-EGFR antibody was obtained from Cell Signaling (USA).

    Techniques:

    Overexpression of TIEG1 attenuates EGFR expression and histone acetylation of the EGFR promoter in MDA-MB-231HM cells. (A and B) TIEG1 expression vector or control vector was transfected into MDA-MB-231HM cells and generated stable transfectants, MDA-MB-231HM/TIEG1

    Journal: Molecular and Cellular Biology

    Article Title: TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway

    doi: 10.1128/MCB.06152-11

    Figure Lengend Snippet: Overexpression of TIEG1 attenuates EGFR expression and histone acetylation of the EGFR promoter in MDA-MB-231HM cells. (A and B) TIEG1 expression vector or control vector was transfected into MDA-MB-231HM cells and generated stable transfectants, MDA-MB-231HM/TIEG1

    Article Snippet: Tissue sections were incubated with monoclonal antibodies against human TIEG1 or EGFR (Santa Cruz, Biotechnology, Santa Cruz, CA) at a dilution of 1:100 overnight at 4°C.

    Techniques: Over Expression, Expressing, Multiple Displacement Amplification, Plasmid Preparation, Transfection, Generated

    TIEG1 inhibits EGFR promoter activity in MDA-MB-231HM and MDA-MB-468 cells. (A) Model depicting binding of TIEG1 transcriptional factor to the Sp1 sites on the EGFR promoter. (B and C) TIEG1 and HDAC1 inhibited EGFR promoter activity in MDA-MB-231HM and

    Journal: Molecular and Cellular Biology

    Article Title: TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway

    doi: 10.1128/MCB.06152-11

    Figure Lengend Snippet: TIEG1 inhibits EGFR promoter activity in MDA-MB-231HM and MDA-MB-468 cells. (A) Model depicting binding of TIEG1 transcriptional factor to the Sp1 sites on the EGFR promoter. (B and C) TIEG1 and HDAC1 inhibited EGFR promoter activity in MDA-MB-231HM and

    Article Snippet: Tissue sections were incubated with monoclonal antibodies against human TIEG1 or EGFR (Santa Cruz, Biotechnology, Santa Cruz, CA) at a dilution of 1:100 overnight at 4°C.

    Techniques: Activity Assay, Multiple Displacement Amplification, Binding Assay

    TIEG1 expression is associated with EGFR expression and metastasis in breast tumors.

    Journal: Molecular and Cellular Biology

    Article Title: TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway

    doi: 10.1128/MCB.06152-11

    Figure Lengend Snippet: TIEG1 expression is associated with EGFR expression and metastasis in breast tumors.

    Article Snippet: Tissue sections were incubated with monoclonal antibodies against human TIEG1 or EGFR (Santa Cruz, Biotechnology, Santa Cruz, CA) at a dilution of 1:100 overnight at 4°C.

    Techniques: Expressing

    Binding status of TIEG1 complex and histone acetylation on the EGFR promoter in MDA-MB-231 and MDA-MB-231HM cells. (A and B) Relative TIEG1 and EGFR mRNA levels were detected in MDA-MB-231 and MDA-MB-231HM cells. Total RNA was extracted from the cells

    Journal: Molecular and Cellular Biology

    Article Title: TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway

    doi: 10.1128/MCB.06152-11

    Figure Lengend Snippet: Binding status of TIEG1 complex and histone acetylation on the EGFR promoter in MDA-MB-231 and MDA-MB-231HM cells. (A and B) Relative TIEG1 and EGFR mRNA levels were detected in MDA-MB-231 and MDA-MB-231HM cells. Total RNA was extracted from the cells

    Article Snippet: Tissue sections were incubated with monoclonal antibodies against human TIEG1 or EGFR (Santa Cruz, Biotechnology, Santa Cruz, CA) at a dilution of 1:100 overnight at 4°C.

    Techniques: Binding Assay, Multiple Displacement Amplification

    TIEG1 is associated with EGFR expression in breast cancer tissues. (A) TIEG1 and EGFR expression in breast cancer tissues. Immunohistochemical staining was carried out on histological sections in 90 pairs of breast cancer tissues with the anti-TIEG1 or

    Journal: Molecular and Cellular Biology

    Article Title: TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway

    doi: 10.1128/MCB.06152-11

    Figure Lengend Snippet: TIEG1 is associated with EGFR expression in breast cancer tissues. (A) TIEG1 and EGFR expression in breast cancer tissues. Immunohistochemical staining was carried out on histological sections in 90 pairs of breast cancer tissues with the anti-TIEG1 or

    Article Snippet: Tissue sections were incubated with monoclonal antibodies against human TIEG1 or EGFR (Santa Cruz, Biotechnology, Santa Cruz, CA) at a dilution of 1:100 overnight at 4°C.

    Techniques: Expressing, Immunohistochemistry, Staining

    TIEG1 inhibits breast cancer cell invasion by inhibition of EGFR signaling pathway. (A) Overexpression of TIEG1 inhibited the EGFR signaling pathway. TIEG1 expression vector or control vector was transfected into MDA-MB-231HM or MDA-MB-468 cells and generated

    Journal: Molecular and Cellular Biology

    Article Title: TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway

    doi: 10.1128/MCB.06152-11

    Figure Lengend Snippet: TIEG1 inhibits breast cancer cell invasion by inhibition of EGFR signaling pathway. (A) Overexpression of TIEG1 inhibited the EGFR signaling pathway. TIEG1 expression vector or control vector was transfected into MDA-MB-231HM or MDA-MB-468 cells and generated

    Article Snippet: Tissue sections were incubated with monoclonal antibodies against human TIEG1 or EGFR (Santa Cruz, Biotechnology, Santa Cruz, CA) at a dilution of 1:100 overnight at 4°C.

    Techniques: Inhibition, Over Expression, Expressing, Plasmid Preparation, Transfection, Multiple Displacement Amplification, Generated

    TIEG1 inhibits breast cancer metastasis by inhibition of EGFR signaling pathway. (A and B) MDA-MB-231HM/TIEG1, MDA-MB-231HM/Vector, MDA-MB-231, MDA-MB-468/TIEG1, and MDA-MB-468/Vector cells, as indicated, were injected into the mammary fat pad of athymic

    Journal: Molecular and Cellular Biology

    Article Title: TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway

    doi: 10.1128/MCB.06152-11

    Figure Lengend Snippet: TIEG1 inhibits breast cancer metastasis by inhibition of EGFR signaling pathway. (A and B) MDA-MB-231HM/TIEG1, MDA-MB-231HM/Vector, MDA-MB-231, MDA-MB-468/TIEG1, and MDA-MB-468/Vector cells, as indicated, were injected into the mammary fat pad of athymic

    Article Snippet: Tissue sections were incubated with monoclonal antibodies against human TIEG1 or EGFR (Santa Cruz, Biotechnology, Santa Cruz, CA) at a dilution of 1:100 overnight at 4°C.

    Techniques: Inhibition, Multiple Displacement Amplification, Plasmid Preparation, Injection

    Knockdown of TIEG1 induces EGFR expression in MDA-MB-231 cells. (A and B) Knockdown of TIEG1 in MDA-MB-231 cells induced EGFR expression. MDA-MB-231 cells were treated with 100 nM TIEG1 siRNA or nontargeting siRNA for 48 h. Real-time PCR (A) and Western

    Journal: Molecular and Cellular Biology

    Article Title: TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway

    doi: 10.1128/MCB.06152-11

    Figure Lengend Snippet: Knockdown of TIEG1 induces EGFR expression in MDA-MB-231 cells. (A and B) Knockdown of TIEG1 in MDA-MB-231 cells induced EGFR expression. MDA-MB-231 cells were treated with 100 nM TIEG1 siRNA or nontargeting siRNA for 48 h. Real-time PCR (A) and Western

    Article Snippet: Tissue sections were incubated with monoclonal antibodies against human TIEG1 or EGFR (Santa Cruz, Biotechnology, Santa Cruz, CA) at a dilution of 1:100 overnight at 4°C.

    Techniques: Expressing, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot

    TIEG1 expression is associated with EGFR expression and metastasis in breast tumors.

    Journal: Molecular and Cellular Biology

    Article Title: TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway

    doi: 10.1128/MCB.06152-11

    Figure Lengend Snippet: TIEG1 expression is associated with EGFR expression and metastasis in breast tumors.

    Article Snippet: Tissue sections were incubated with monoclonal antibodies against human TIEG1 or EGFR (Santa Cruz, Biotechnology, Santa Cruz, CA) at a dilution of 1:100 overnight at 4°C.

    Techniques: Expressing

    Formation of the EGFR-Grb2–PI3K-C2β complex in vitro. Recombinant EE-tagged PI3K-C2β (100 ng) was incubated in lysis buffer for 2 h at 4°C in the absence or presence of Grb2-GST fusion protein (100 ng) or GST. Immobilized EGFR, isolated by immunoprecipitation (Ab1; Oncogene Science) from lysates of confluent and quiescent cultures of A431 cells, was phosphorylated for 30 min at 30°C in protein kinase buffer upon addition of ATP. Either phosphorylated EGFR, nonphosphorylated EGFR, or mock immunoprecipitate was added to the Grb2–PI3K-C2β sample, and the incubation was continued for a further 1 h. Beads containing immobilized receptor and associated proteins were isolated by centrifugation, washed, fractionated by SDS-PAGE, and Western blotted with either anti-EGFR antibody, antiphosphotyrosine, anti-Grb2, or anti-PI3K-C2β antibody. PY, phosphotyrosine.

    Journal: Molecular and Cellular Biology

    Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

    doi: 10.1128/MCB.21.19.6660-6667.2001

    Figure Lengend Snippet: Formation of the EGFR-Grb2–PI3K-C2β complex in vitro. Recombinant EE-tagged PI3K-C2β (100 ng) was incubated in lysis buffer for 2 h at 4°C in the absence or presence of Grb2-GST fusion protein (100 ng) or GST. Immobilized EGFR, isolated by immunoprecipitation (Ab1; Oncogene Science) from lysates of confluent and quiescent cultures of A431 cells, was phosphorylated for 30 min at 30°C in protein kinase buffer upon addition of ATP. Either phosphorylated EGFR, nonphosphorylated EGFR, or mock immunoprecipitate was added to the Grb2–PI3K-C2β sample, and the incubation was continued for a further 1 h. Beads containing immobilized receptor and associated proteins were isolated by centrifugation, washed, fractionated by SDS-PAGE, and Western blotted with either anti-EGFR antibody, antiphosphotyrosine, anti-Grb2, or anti-PI3K-C2β antibody. PY, phosphotyrosine.

    Article Snippet: After isolation and washing, the resultant immune complexes were extracted, fractionated by SDS-PAGE, and Western blotted with antisera to either PI3K-C2β (Fig. , upper panels), SOS1/2 (Santa Cruz) (middle panels), or anti-EGFR (Santa Cruz) (lower panels).

    Techniques: In Vitro, Recombinant, Incubation, Lysis, Isolation, Immunoprecipitation, Centrifugation, SDS Page, Western Blot

    Grb2 increases the catalytic activity of PI3K-C2β. Aliquots of recombinant EE-tagged PI3K-C2β (100 ng) were incubated in PI3K assay buffer for 2 h at 4°C with either Grb2-GST fusion protein (100 ng) or GST. After this time, immobilized EGFR previously immunoprecipitated (Ab-1; Oncogene Science) from lysates of quiescent A431 cells and autophosphorylated in vitro or mock control was added. Incubation was continued for a further 1 h. Following addition of kinase buffer, phosphatidylinositol (200 μg/ml) and [γ- 32 P]ATP, samples (50 μl) were incubated at room temperature for 20 min. Radiolabeled phosphoinositides were extracted and aliquots were fractionated by thin-layer chromatography and visualized by autoradiography. Representative data are shown in the upper panel. Quantification of PtdIns3P was undertaken by scanning densitometry. In the lower panel, data are displayed as means ± standard errors of the means ( n = 6). Under the conditions used, all reactions displayed linear kinetics.

    Journal: Molecular and Cellular Biology

    Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

    doi: 10.1128/MCB.21.19.6660-6667.2001

    Figure Lengend Snippet: Grb2 increases the catalytic activity of PI3K-C2β. Aliquots of recombinant EE-tagged PI3K-C2β (100 ng) were incubated in PI3K assay buffer for 2 h at 4°C with either Grb2-GST fusion protein (100 ng) or GST. After this time, immobilized EGFR previously immunoprecipitated (Ab-1; Oncogene Science) from lysates of quiescent A431 cells and autophosphorylated in vitro or mock control was added. Incubation was continued for a further 1 h. Following addition of kinase buffer, phosphatidylinositol (200 μg/ml) and [γ- 32 P]ATP, samples (50 μl) were incubated at room temperature for 20 min. Radiolabeled phosphoinositides were extracted and aliquots were fractionated by thin-layer chromatography and visualized by autoradiography. Representative data are shown in the upper panel. Quantification of PtdIns3P was undertaken by scanning densitometry. In the lower panel, data are displayed as means ± standard errors of the means ( n = 6). Under the conditions used, all reactions displayed linear kinetics.

    Article Snippet: After isolation and washing, the resultant immune complexes were extracted, fractionated by SDS-PAGE, and Western blotted with antisera to either PI3K-C2β (Fig. , upper panels), SOS1/2 (Santa Cruz) (middle panels), or anti-EGFR (Santa Cruz) (lower panels).

    Techniques: Activity Assay, Recombinant, Incubation, Immunoprecipitation, In Vitro, Thin Layer Chromatography, Autoradiography

    The N terminus of PI3K-C2β but not PI3K-C2α interacts with the EGFR. A431 cells were incubated in the absence (−) or presence (+) of EGF (100 nM) for 10 min. Cell lysates were prepared and incubated with either GST, GST–PI3K-C2α(2-345), GST–PI3K-C2β(2-298), or GST–PI3K-C2α(1549-1686) for 4 h at 4°C. Fusion protein was isolated using glutathione-Sepharose beads, and associated proteins were fractionated by SDS-PAGE. Membranes were Western blotted with antiphosphotyrosine antibody (upper panel) or anti-EGFR antibody (lower panel). Arrows indicate the EGFR (approximately 180 kDa) and associated phosphoproteins. PY, phosphotyrosine.

    Journal: Molecular and Cellular Biology

    Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

    doi: 10.1128/MCB.21.19.6660-6667.2001

    Figure Lengend Snippet: The N terminus of PI3K-C2β but not PI3K-C2α interacts with the EGFR. A431 cells were incubated in the absence (−) or presence (+) of EGF (100 nM) for 10 min. Cell lysates were prepared and incubated with either GST, GST–PI3K-C2α(2-345), GST–PI3K-C2β(2-298), or GST–PI3K-C2α(1549-1686) for 4 h at 4°C. Fusion protein was isolated using glutathione-Sepharose beads, and associated proteins were fractionated by SDS-PAGE. Membranes were Western blotted with antiphosphotyrosine antibody (upper panel) or anti-EGFR antibody (lower panel). Arrows indicate the EGFR (approximately 180 kDa) and associated phosphoproteins. PY, phosphotyrosine.

    Article Snippet: After isolation and washing, the resultant immune complexes were extracted, fractionated by SDS-PAGE, and Western blotted with antisera to either PI3K-C2β (Fig. , upper panels), SOS1/2 (Santa Cruz) (middle panels), or anti-EGFR (Santa Cruz) (lower panels).

    Techniques: Incubation, Isolation, SDS Page, Western Blot

    PI3K-C2β(2-298) affinity purifies EGFR, Shc, and Grb2 in a dose-dependent manner. Lysates from EGF-stimulated A431 cells were incubated with various additions of recombinant GST–PI3K-C2β(2-298) protein as shown. After 4 h at 4°C the fusion and associated proteins were isolated by centrifugation and were washed. They were then extracted, fractionated by SDS-PAGE, and Western blotted with antiphosphotyrosine (upper panel), anti-Shc (middle panel), or anti-Grb2 (lower panel). PY, phosphotyrosine.

    Journal: Molecular and Cellular Biology

    Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

    doi: 10.1128/MCB.21.19.6660-6667.2001

    Figure Lengend Snippet: PI3K-C2β(2-298) affinity purifies EGFR, Shc, and Grb2 in a dose-dependent manner. Lysates from EGF-stimulated A431 cells were incubated with various additions of recombinant GST–PI3K-C2β(2-298) protein as shown. After 4 h at 4°C the fusion and associated proteins were isolated by centrifugation and were washed. They were then extracted, fractionated by SDS-PAGE, and Western blotted with antiphosphotyrosine (upper panel), anti-Shc (middle panel), or anti-Grb2 (lower panel). PY, phosphotyrosine.

    Article Snippet: After isolation and washing, the resultant immune complexes were extracted, fractionated by SDS-PAGE, and Western blotted with antisera to either PI3K-C2β (Fig. , upper panels), SOS1/2 (Santa Cruz) (middle panels), or anti-EGFR (Santa Cruz) (lower panels).

    Techniques: Incubation, Recombinant, Isolation, Centrifugation, SDS Page, Western Blot

    Polyproline-rich peptides competitively attenuate the association of PI3K-C2β with the EGFR. Peptides (25 μM) corresponding to each of the three PI3K-C2β polyproline-rich regions at residues 127 to 140 (KKLSPPPLPPRASI), 140 to 153 (IWDTPPLPPRKGSP) and 252 to 265 (SKTMPPQVPPRTYA) or control residues 69 to 82 (NSLSPLEGPPNHST) were added to lysates of EGF-stimulated A431 cells. Samples were incubated at 4°C, and recombinant PI3K-C2β(2-298) (5 μg) was added 30 min later together with glutathione-Sepharose beads. After 4 h at 4°C, fusion protein was isolated by centrifugation, fractionated by SDS-PAGE, and Western blotted with antiphosphotyrosine (upper panel) or anti-PI3KC2β antibody (lower panel). PY, phosphotyrosine.

    Journal: Molecular and Cellular Biology

    Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

    doi: 10.1128/MCB.21.19.6660-6667.2001

    Figure Lengend Snippet: Polyproline-rich peptides competitively attenuate the association of PI3K-C2β with the EGFR. Peptides (25 μM) corresponding to each of the three PI3K-C2β polyproline-rich regions at residues 127 to 140 (KKLSPPPLPPRASI), 140 to 153 (IWDTPPLPPRKGSP) and 252 to 265 (SKTMPPQVPPRTYA) or control residues 69 to 82 (NSLSPLEGPPNHST) were added to lysates of EGF-stimulated A431 cells. Samples were incubated at 4°C, and recombinant PI3K-C2β(2-298) (5 μg) was added 30 min later together with glutathione-Sepharose beads. After 4 h at 4°C, fusion protein was isolated by centrifugation, fractionated by SDS-PAGE, and Western blotted with antiphosphotyrosine (upper panel) or anti-PI3KC2β antibody (lower panel). PY, phosphotyrosine.

    Article Snippet: After isolation and washing, the resultant immune complexes were extracted, fractionated by SDS-PAGE, and Western blotted with antisera to either PI3K-C2β (Fig. , upper panels), SOS1/2 (Santa Cruz) (middle panels), or anti-EGFR (Santa Cruz) (lower panels).

    Techniques: Incubation, Recombinant, Isolation, Centrifugation, SDS Page, Western Blot

    N-terminal fragments of PI3K-C2β establish that two proline-rich motifs are required for association with EGFR. A series of GST fusion proteins representing residues 2 to 130, 2 to 143, 2 to 157, 2 to 255, and 2 to 298 were incubated for 4 h at 4°C with lysates of A431 cells that had been treated in the absence (−) or presence (+) of EGF (100 nM) for 10 min. For control, EGFR was also immunoprecipitated from these lysates (Ab1; Oncogene Science). Each fusion and associated protein was isolated using glutathione-Sepharose beads, washed, fractionated by SDS-PAGE, and Western blotted with antiphosphotyrosine antibody to visualize the activated EGFR (upper panel) and anti-Grb2 antibody (lower panel). PY, phosphotyrosine.

    Journal: Molecular and Cellular Biology

    Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

    doi: 10.1128/MCB.21.19.6660-6667.2001

    Figure Lengend Snippet: N-terminal fragments of PI3K-C2β establish that two proline-rich motifs are required for association with EGFR. A series of GST fusion proteins representing residues 2 to 130, 2 to 143, 2 to 157, 2 to 255, and 2 to 298 were incubated for 4 h at 4°C with lysates of A431 cells that had been treated in the absence (−) or presence (+) of EGF (100 nM) for 10 min. For control, EGFR was also immunoprecipitated from these lysates (Ab1; Oncogene Science). Each fusion and associated protein was isolated using glutathione-Sepharose beads, washed, fractionated by SDS-PAGE, and Western blotted with antiphosphotyrosine antibody to visualize the activated EGFR (upper panel) and anti-Grb2 antibody (lower panel). PY, phosphotyrosine.

    Article Snippet: After isolation and washing, the resultant immune complexes were extracted, fractionated by SDS-PAGE, and Western blotted with antisera to either PI3K-C2β (Fig. , upper panels), SOS1/2 (Santa Cruz) (middle panels), or anti-EGFR (Santa Cruz) (lower panels).

    Techniques: Incubation, Immunoprecipitation, Isolation, SDS Page, Western Blot

    Isolation of PI3K-C2β, SOS, and EGFR in anti-Grb2 immunoprecipitates (ippt). Confluent and quiescent cultures of human renal epithelial cells (HKC-8) and breast cancer cells (HTM3551, T47D, and SKBr3) were incubated with (+) or without (−) EGF (100 nM) for 10 min. Lysates were prepared to which anti-Grb2 antibody (Santa Cruz C-23) was added for 4 h at 4°C. Immune complexes were isolated following addition of protein A-Sepharose and centrifugation. Proteins were extracted, fractionated by SDS-PAGE, and Western blotted with antisera to PI3K-C2β (upper panels), SOS 1/2 (D21; Santa Cruz) (middle panels), and EGFR (1005; Santa Cruz) (lower panels).

    Journal: Molecular and Cellular Biology

    Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

    doi: 10.1128/MCB.21.19.6660-6667.2001

    Figure Lengend Snippet: Isolation of PI3K-C2β, SOS, and EGFR in anti-Grb2 immunoprecipitates (ippt). Confluent and quiescent cultures of human renal epithelial cells (HKC-8) and breast cancer cells (HTM3551, T47D, and SKBr3) were incubated with (+) or without (−) EGF (100 nM) for 10 min. Lysates were prepared to which anti-Grb2 antibody (Santa Cruz C-23) was added for 4 h at 4°C. Immune complexes were isolated following addition of protein A-Sepharose and centrifugation. Proteins were extracted, fractionated by SDS-PAGE, and Western blotted with antisera to PI3K-C2β (upper panels), SOS 1/2 (D21; Santa Cruz) (middle panels), and EGFR (1005; Santa Cruz) (lower panels).

    Article Snippet: After isolation and washing, the resultant immune complexes were extracted, fractionated by SDS-PAGE, and Western blotted with antisera to either PI3K-C2β (Fig. , upper panels), SOS1/2 (Santa Cruz) (middle panels), or anti-EGFR (Santa Cruz) (lower panels).

    Techniques: Isolation, Incubation, Centrifugation, SDS Page, Western Blot

    EGFR co-immunoprecipitates with flotillins. HeLa cells (serum-grown, unstimulated, or EGF stimulated) were lysed and immunoprecipitated with antibodies against flot-2. Co-immunoprecipitation of EGFR with flotillins was detected in all samples. After 5 min of EGF, a precipitated band (185 kDa) migrated slightly higher than EGFR (180 kDa) in unstimulated samples and corresponded to the Tyr-1173-phosphorylated EGFR, as evidenced by detection with an anti-Tyr(P)-1173 antibody. Flot-1 (47 kDa) coprecipitated with flot-2 (48 kDa) in all samples, whereas no precipitation of EGFR or flotillins was detected in the control immunoprecipitation carried out with an anti-myc antibody ( Control IP ). Equal loading was verified with GAPDH (37 kDa).

    Journal: The Journal of Biological Chemistry

    Article Title: Flotillin-1/Reggie-2 Protein Plays Dual Role in Activation of Receptor-tyrosine Kinase/Mitogen-activated Protein Kinase Signaling *

    doi: 10.1074/jbc.M111.287599

    Figure Lengend Snippet: EGFR co-immunoprecipitates with flotillins. HeLa cells (serum-grown, unstimulated, or EGF stimulated) were lysed and immunoprecipitated with antibodies against flot-2. Co-immunoprecipitation of EGFR with flotillins was detected in all samples. After 5 min of EGF, a precipitated band (185 kDa) migrated slightly higher than EGFR (180 kDa) in unstimulated samples and corresponded to the Tyr-1173-phosphorylated EGFR, as evidenced by detection with an anti-Tyr(P)-1173 antibody. Flot-1 (47 kDa) coprecipitated with flot-2 (48 kDa) in all samples, whereas no precipitation of EGFR or flotillins was detected in the control immunoprecipitation carried out with an anti-myc antibody ( Control IP ). Equal loading was verified with GAPDH (37 kDa).

    Article Snippet: The cells were either stimulated for 2 min with 100 ng/ml EGF or not, fixed, and labeled with an anti-EGFR antibody (1:100, Santa Cruz) and subsequently with a Cy3-conjugated secondary antibody.

    Techniques: Immunoprecipitation

    Flot-1 depletion does not inhibit EGFR endocytosis and ubiquitination. F1-KD and control cells were incubated with 125 I-EGF (1.5 ng/ml) for the indicated time points, and the internalized versus cell surface-bound EGFs were measured. The data are a summary of three independent experiments, each with triplicate samples for each time point. A , the graph shows the ratio of internalized to surface bound EGF as a function of endocytosis time, including the S.D. The difference between control cells and F1-KD cells was not significant ( ns. ). B , F1-KD efficiency in the cells used for the experiment is shown. C , Flot-1 was depleted by means of RNAi in HeLa cells that were subsequently starved and stimulated or not with EGF for 2, 10, or 30 min. EGFR was immunoprecipitated ( IP ) from the cells, and the precipitates were probed with an anti-ubiquitin antibody. IgG , an isotype matched control antibody was used to show the specificity. D , quantification of EGFR ubiquitination from four individual experiments is shown. Ubiquitin signals were normalized to total EGFR. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Flotillin-1/Reggie-2 Protein Plays Dual Role in Activation of Receptor-tyrosine Kinase/Mitogen-activated Protein Kinase Signaling *

    doi: 10.1074/jbc.M111.287599

    Figure Lengend Snippet: Flot-1 depletion does not inhibit EGFR endocytosis and ubiquitination. F1-KD and control cells were incubated with 125 I-EGF (1.5 ng/ml) for the indicated time points, and the internalized versus cell surface-bound EGFs were measured. The data are a summary of three independent experiments, each with triplicate samples for each time point. A , the graph shows the ratio of internalized to surface bound EGF as a function of endocytosis time, including the S.D. The difference between control cells and F1-KD cells was not significant ( ns. ). B , F1-KD efficiency in the cells used for the experiment is shown. C , Flot-1 was depleted by means of RNAi in HeLa cells that were subsequently starved and stimulated or not with EGF for 2, 10, or 30 min. EGFR was immunoprecipitated ( IP ) from the cells, and the precipitates were probed with an anti-ubiquitin antibody. IgG , an isotype matched control antibody was used to show the specificity. D , quantification of EGFR ubiquitination from four individual experiments is shown. Ubiquitin signals were normalized to total EGFR. *, p

    Article Snippet: The cells were either stimulated for 2 min with 100 ng/ml EGF or not, fixed, and labeled with an anti-EGFR antibody (1:100, Santa Cruz) and subsequently with a Cy3-conjugated secondary antibody.

    Techniques: Incubation, Immunoprecipitation

    EGF-induced clustering of EGFR at the cell surface is impaired by flot-1 knockdown. Flot-1 was knocked down in HeLa cells by siRNA. Knockdown and control cells were starved and stimulated or not for 2 min with EGF. The cells were fixed and immunostained for EGF receptor. Cell surface associated EGFR was detected by total internal reflection microscopy. The images ( A ) were analyzed for the mean EGFR cluster intensity. B , shown is quantification of the EGFR cluster intensity upon EGF stimulation in control and F1-KD cells ( n = 3). ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Flotillin-1/Reggie-2 Protein Plays Dual Role in Activation of Receptor-tyrosine Kinase/Mitogen-activated Protein Kinase Signaling *

    doi: 10.1074/jbc.M111.287599

    Figure Lengend Snippet: EGF-induced clustering of EGFR at the cell surface is impaired by flot-1 knockdown. Flot-1 was knocked down in HeLa cells by siRNA. Knockdown and control cells were starved and stimulated or not for 2 min with EGF. The cells were fixed and immunostained for EGF receptor. Cell surface associated EGFR was detected by total internal reflection microscopy. The images ( A ) were analyzed for the mean EGFR cluster intensity. B , shown is quantification of the EGFR cluster intensity upon EGF stimulation in control and F1-KD cells ( n = 3). ***, p

    Article Snippet: The cells were either stimulated for 2 min with 100 ng/ml EGF or not, fixed, and labeled with an anti-EGFR antibody (1:100, Santa Cruz) and subsequently with a Cy3-conjugated secondary antibody.

    Techniques: Microscopy

    Knockdown of flot-1 results in reduced Tyr phosphorylation of EGFR. Flot-1 was depleted by means of siRNAs. The cells were subsequently starved and stimulated or not with EGF for 2, 10, or 30 min, and EGFR was immunoprecipitated ( IP ). Precipitated proteins were separated by SDS-PAGE and probed with the indicated antibodies. The cell lysates were analyzed with the monoclonal antibody against flot-1 to verify its depletion, and GAPDH was used as an equal loading control. Effect of flotillin knockdown on total tyrosine phosphorylation ( pY ; A ) and Tyr-1173 ( B ) was measured. C , the bar graph show a densitometric quantification of the phosphorylation of Tyr-1173. The signals of phosphorylated Tyr-1173 were normalized to the amount of total EGFR. Bars represent the mean ± S.D. of at least three individual experiments. ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Flotillin-1/Reggie-2 Protein Plays Dual Role in Activation of Receptor-tyrosine Kinase/Mitogen-activated Protein Kinase Signaling *

    doi: 10.1074/jbc.M111.287599

    Figure Lengend Snippet: Knockdown of flot-1 results in reduced Tyr phosphorylation of EGFR. Flot-1 was depleted by means of siRNAs. The cells were subsequently starved and stimulated or not with EGF for 2, 10, or 30 min, and EGFR was immunoprecipitated ( IP ). Precipitated proteins were separated by SDS-PAGE and probed with the indicated antibodies. The cell lysates were analyzed with the monoclonal antibody against flot-1 to verify its depletion, and GAPDH was used as an equal loading control. Effect of flotillin knockdown on total tyrosine phosphorylation ( pY ; A ) and Tyr-1173 ( B ) was measured. C , the bar graph show a densitometric quantification of the phosphorylation of Tyr-1173. The signals of phosphorylated Tyr-1173 were normalized to the amount of total EGFR. Bars represent the mean ± S.D. of at least three individual experiments. ***, p

    Article Snippet: The cells were either stimulated for 2 min with 100 ng/ml EGF or not, fixed, and labeled with an anti-EGFR antibody (1:100, Santa Cruz) and subsequently with a Cy3-conjugated secondary antibody.

    Techniques: Immunoprecipitation, SDS Page

    EGFR and Grb2 do not translocate into light fractions in F1-KD cells. Flot-1 was knocked down in HeLa cells by siRNA. The cells were starved and stimulated with 100 ng/ml EGF for 30 min on ice or left untreated. A , rafts were isolated from the cells using a detergent-free method. Fractions were collected from the top (fraction 1 = the lightest fraction), run on SDS-PAGE, and analyzed by Western blotting. The localization of EGFR (180 kDa), Grb2 (25 kDa), CRAF (74 kDa), flotillins (47/48 kDa), and GAPDH (37 kDa) was studied. The raft marker GM1 was detected with HRP-coupled cholera toxin in a slot blot. Quantification of the relative distribution in the light fractions: EGFR ( B ); Grb2 ( C ); CRAF ( D ); flotillin-2 ( E ) (summary of four independent experiments). CT-B , cholera toxin B subunit. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Flotillin-1/Reggie-2 Protein Plays Dual Role in Activation of Receptor-tyrosine Kinase/Mitogen-activated Protein Kinase Signaling *

    doi: 10.1074/jbc.M111.287599

    Figure Lengend Snippet: EGFR and Grb2 do not translocate into light fractions in F1-KD cells. Flot-1 was knocked down in HeLa cells by siRNA. The cells were starved and stimulated with 100 ng/ml EGF for 30 min on ice or left untreated. A , rafts were isolated from the cells using a detergent-free method. Fractions were collected from the top (fraction 1 = the lightest fraction), run on SDS-PAGE, and analyzed by Western blotting. The localization of EGFR (180 kDa), Grb2 (25 kDa), CRAF (74 kDa), flotillins (47/48 kDa), and GAPDH (37 kDa) was studied. The raft marker GM1 was detected with HRP-coupled cholera toxin in a slot blot. Quantification of the relative distribution in the light fractions: EGFR ( B ); Grb2 ( C ); CRAF ( D ); flotillin-2 ( E ) (summary of four independent experiments). CT-B , cholera toxin B subunit. *, p

    Article Snippet: The cells were either stimulated for 2 min with 100 ng/ml EGF or not, fixed, and labeled with an anti-EGFR antibody (1:100, Santa Cruz) and subsequently with a Cy3-conjugated secondary antibody.

    Techniques: Isolation, SDS Page, Western Blot, Marker, Dot Blot

    RHOB induces resistance to erlotinib through the AKT pathway HCC4006, HCC827, HCC2935, and H3255 cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 4 h with erlotinib at concentrations corresponding to the respective IC 50 values determined for each control cell line. The phosphorylation status of AKT, ERK1/2, and EGFR was assessed by Western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by Western blotting. Representative immunostaining of phospho‐AKT (Ser473) and phospho‐ERK1/2 and their total protein amounts in lung tumors from EGFR L858R / Rhob − / − or EGFR L858R / Rhob +/+ mice treated or not with erlotinib (12.5 mg/kg/day) for 4 days. The remaining hyperplastic areas were selected in erlotinib‐treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bars: 100 μm. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKT myr , myristoylated) or an empty vector (ø) and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay, and AKT overexpression and phosphorylation at Ser473 were assessed by Western blotting. Data are representative of at least three independent experiments. Data are expressed as mean ± SEM from three independent experiments. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: The RAS‐related GTPase RHOB confers resistance to EGFR‐tyrosine kinase inhibitors in non‐small‐cell lung cancer via an AKT‐dependent mechanism

    doi: 10.15252/emmm.201606646

    Figure Lengend Snippet: RHOB induces resistance to erlotinib through the AKT pathway HCC4006, HCC827, HCC2935, and H3255 cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 4 h with erlotinib at concentrations corresponding to the respective IC 50 values determined for each control cell line. The phosphorylation status of AKT, ERK1/2, and EGFR was assessed by Western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by Western blotting. Representative immunostaining of phospho‐AKT (Ser473) and phospho‐ERK1/2 and their total protein amounts in lung tumors from EGFR L858R / Rhob − / − or EGFR L858R / Rhob +/+ mice treated or not with erlotinib (12.5 mg/kg/day) for 4 days. The remaining hyperplastic areas were selected in erlotinib‐treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bars: 100 μm. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKT myr , myristoylated) or an empty vector (ø) and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay, and AKT overexpression and phosphorylation at Ser473 were assessed by Western blotting. Data are representative of at least three independent experiments. Data are expressed as mean ± SEM from three independent experiments. Source data are available online for this figure.

    Article Snippet: Western blot analysis Cell extracts were analyzed by Western blotting with primary antibodies against RHOB, ERK1/2 (Santa Cruz Biotechnology), p‐ERK (T202/Y204), p‐AKT (S473), AKT, pGSK3 (S9), GSK3, p‐EGFR (T1173), EGFR, PARP, cleaved caspase‐3, caspase‐3 (Cell Signaling Technology), or actin (Chemicon).

    Techniques: Transduction, Western Blot, Over Expression, Immunostaining, Mouse Assay, Transfection, Plasmid Preparation, Mutagenesis, MTS Assay

    RHOB loss of expression increases sensitivity to erlotinib in mice with EGFR L858R ‐driven lung tumors Representative H E staining of whole lungs from EGFR L858R / Rhob − / − , EGFR L858R / Rhob +/− , and EGFR L858R / Rhob +/+ mice treated or not with erlotinib (12.5 mg/kg/day) for 4 days. Scale bars: 5 mm. Quantification of the tumor/lung ratio. n = 7 for each group except for EGFR L858R / Rhob +/− placebo ( n = 6). Representative Ki67 immunostaining of EGFR L858R / Rhob − / − , EGFR L858R / Rhob +/− , and EGFR L858R / Rhob +/+ mice treated or not with erlotinib (12.5 mg/kg/day) for 4 days (scale bars: 50 μm), and the corresponding quantification (D). Three independent zones per mouse lung were used for quantification. n = 21 (seven mice) for each group except for EGFR L858R / Rhob +/− placebo ( n = 18; six mice). Immunostaining of cleaved caspase‐3 in lung tumors from EGFR L858R / Rhob +/+ or EGFR L858R / Rhob − / − mice treated for 24 h with erlotinib at 12.5 mg/kg. Black arrows point apoptotic cells. Scale bars: 50 μm. Data information: ** P

    Journal: EMBO Molecular Medicine

    Article Title: The RAS‐related GTPase RHOB confers resistance to EGFR‐tyrosine kinase inhibitors in non‐small‐cell lung cancer via an AKT‐dependent mechanism

    doi: 10.15252/emmm.201606646

    Figure Lengend Snippet: RHOB loss of expression increases sensitivity to erlotinib in mice with EGFR L858R ‐driven lung tumors Representative H E staining of whole lungs from EGFR L858R / Rhob − / − , EGFR L858R / Rhob +/− , and EGFR L858R / Rhob +/+ mice treated or not with erlotinib (12.5 mg/kg/day) for 4 days. Scale bars: 5 mm. Quantification of the tumor/lung ratio. n = 7 for each group except for EGFR L858R / Rhob +/− placebo ( n = 6). Representative Ki67 immunostaining of EGFR L858R / Rhob − / − , EGFR L858R / Rhob +/− , and EGFR L858R / Rhob +/+ mice treated or not with erlotinib (12.5 mg/kg/day) for 4 days (scale bars: 50 μm), and the corresponding quantification (D). Three independent zones per mouse lung were used for quantification. n = 21 (seven mice) for each group except for EGFR L858R / Rhob +/− placebo ( n = 18; six mice). Immunostaining of cleaved caspase‐3 in lung tumors from EGFR L858R / Rhob +/+ or EGFR L858R / Rhob − / − mice treated for 24 h with erlotinib at 12.5 mg/kg. Black arrows point apoptotic cells. Scale bars: 50 μm. Data information: ** P

    Article Snippet: Western blot analysis Cell extracts were analyzed by Western blotting with primary antibodies against RHOB, ERK1/2 (Santa Cruz Biotechnology), p‐ERK (T202/Y204), p‐AKT (S473), AKT, pGSK3 (S9), GSK3, p‐EGFR (T1173), EGFR, PARP, cleaved caspase‐3, caspase‐3 (Cell Signaling Technology), or actin (Chemicon).

    Techniques: Expressing, Mouse Assay, Staining, Immunostaining

    G594 prevents GSK3β phosphorylation in RHOB‐overexpressing cells treated with erlotinib and reverses RHOB‐induced resistance H3255 (A), HCC2935 (B), and HCC827 (C) cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 4 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2, and EGFR (Tyr1173) was assessed by Western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by Western blotting. H3255 (D), HCC2935 (E), or HCC827 (F) cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay. Data are expressed as mean ± SEM from three independent experiments. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: The RAS‐related GTPase RHOB confers resistance to EGFR‐tyrosine kinase inhibitors in non‐small‐cell lung cancer via an AKT‐dependent mechanism

    doi: 10.15252/emmm.201606646

    Figure Lengend Snippet: G594 prevents GSK3β phosphorylation in RHOB‐overexpressing cells treated with erlotinib and reverses RHOB‐induced resistance H3255 (A), HCC2935 (B), and HCC827 (C) cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 4 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2, and EGFR (Tyr1173) was assessed by Western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by Western blotting. H3255 (D), HCC2935 (E), or HCC827 (F) cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay. Data are expressed as mean ± SEM from three independent experiments. Source data are available online for this figure.

    Article Snippet: Western blot analysis Cell extracts were analyzed by Western blotting with primary antibodies against RHOB, ERK1/2 (Santa Cruz Biotechnology), p‐ERK (T202/Y204), p‐AKT (S473), AKT, pGSK3 (S9), GSK3, p‐EGFR (T1173), EGFR, PARP, cleaved caspase‐3, caspase‐3 (Cell Signaling Technology), or actin (Chemicon).

    Techniques: Transduction, Western Blot, Over Expression, MTS Assay

    RHOB overexpression does not affect response to erlotinib in EGFR WT cell lines A549 or H1299 cells were transduced with control (AdCont) or RHOB‐overexpressing adenoviruses (AdRHOB) and treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells. Data are expressed as mean ± SEM from three independent experiments. A549 and H1299 cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 4 h with erlotinib at 1 μM. The phosphorylation status of AKT, ERK1/2, and EGFR was assessed by Western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by Western blotting. EGFR‐mutated HCC4006 cells were used to monitor erlotinib efficiency (right panel). Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: The RAS‐related GTPase RHOB confers resistance to EGFR‐tyrosine kinase inhibitors in non‐small‐cell lung cancer via an AKT‐dependent mechanism

    doi: 10.15252/emmm.201606646

    Figure Lengend Snippet: RHOB overexpression does not affect response to erlotinib in EGFR WT cell lines A549 or H1299 cells were transduced with control (AdCont) or RHOB‐overexpressing adenoviruses (AdRHOB) and treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells. Data are expressed as mean ± SEM from three independent experiments. A549 and H1299 cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 4 h with erlotinib at 1 μM. The phosphorylation status of AKT, ERK1/2, and EGFR was assessed by Western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by Western blotting. EGFR‐mutated HCC4006 cells were used to monitor erlotinib efficiency (right panel). Source data are available online for this figure.

    Article Snippet: Western blot analysis Cell extracts were analyzed by Western blotting with primary antibodies against RHOB, ERK1/2 (Santa Cruz Biotechnology), p‐ERK (T202/Y204), p‐AKT (S473), AKT, pGSK3 (S9), GSK3, p‐EGFR (T1173), EGFR, PARP, cleaved caspase‐3, caspase‐3 (Cell Signaling Technology), or actin (Chemicon).

    Techniques: Over Expression, Transduction, MTS Assay, Western Blot

    The AKT inhibitor G594 resensitizes EGFR L858R mouse tumors to erlotinib Representative H E staining of lung tumors from EGFR L858R / Rhob − / − , EGFR L858R / Rhob +/− , and EGFR L858R / Rhob +/+ mice treated or not during 4 days with erlotinib (12.5 mg/kg/day), the AKT inhibitor G594 (25 mg/kg/day), or a combination of both drugs. Scale bars: 500 μm. Quantification of the tumor/lung ratio of mice treated or not with the individual drugs or with a combination of both. Representative Ki67 immunostaining of lung tumors from EGFR L858R / Rhob −/− , EGFR L858R / Rhob +/− , and EGFR L858R / Rhob +/+ mice treated or not with erlotinib alone (12.5 mg/kg/day) or in combination with the AKT inhibitor G594 (25 mg/kg/day) for 4 days (scale bars: 50 μm), and the corresponding quantification (D). Data information: ** P

    Journal: EMBO Molecular Medicine

    Article Title: The RAS‐related GTPase RHOB confers resistance to EGFR‐tyrosine kinase inhibitors in non‐small‐cell lung cancer via an AKT‐dependent mechanism

    doi: 10.15252/emmm.201606646

    Figure Lengend Snippet: The AKT inhibitor G594 resensitizes EGFR L858R mouse tumors to erlotinib Representative H E staining of lung tumors from EGFR L858R / Rhob − / − , EGFR L858R / Rhob +/− , and EGFR L858R / Rhob +/+ mice treated or not during 4 days with erlotinib (12.5 mg/kg/day), the AKT inhibitor G594 (25 mg/kg/day), or a combination of both drugs. Scale bars: 500 μm. Quantification of the tumor/lung ratio of mice treated or not with the individual drugs or with a combination of both. Representative Ki67 immunostaining of lung tumors from EGFR L858R / Rhob −/− , EGFR L858R / Rhob +/− , and EGFR L858R / Rhob +/+ mice treated or not with erlotinib alone (12.5 mg/kg/day) or in combination with the AKT inhibitor G594 (25 mg/kg/day) for 4 days (scale bars: 50 μm), and the corresponding quantification (D). Data information: ** P

    Article Snippet: Western blot analysis Cell extracts were analyzed by Western blotting with primary antibodies against RHOB, ERK1/2 (Santa Cruz Biotechnology), p‐ERK (T202/Y204), p‐AKT (S473), AKT, pGSK3 (S9), GSK3, p‐EGFR (T1173), EGFR, PARP, cleaved caspase‐3, caspase‐3 (Cell Signaling Technology), or actin (Chemicon).

    Techniques: Staining, Mouse Assay, Immunostaining

    AKT inhibition sensitizes RHOB‐expressing cells to erlotinib HCC4006 cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 4 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2, and EGFR (Tyr1173) was assessed by Western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by Western blotting. HCC4006 cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay. Data are expressed as mean ± SEM from three independent experiments. HCC4006 cells were transduced with control (AdCont) or RHOB‐overexpressing adenoviruses (AdRHOB) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594. The surviving cell fraction was determined by an MTS assay, and erlotinib IC 50 values were determined for each condition (** P

    Journal: EMBO Molecular Medicine

    Article Title: The RAS‐related GTPase RHOB confers resistance to EGFR‐tyrosine kinase inhibitors in non‐small‐cell lung cancer via an AKT‐dependent mechanism

    doi: 10.15252/emmm.201606646

    Figure Lengend Snippet: AKT inhibition sensitizes RHOB‐expressing cells to erlotinib HCC4006 cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 4 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2, and EGFR (Tyr1173) was assessed by Western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by Western blotting. HCC4006 cells were transduced with control (AdCont) or RHOB‐overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay. Data are expressed as mean ± SEM from three independent experiments. HCC4006 cells were transduced with control (AdCont) or RHOB‐overexpressing adenoviruses (AdRHOB) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594. The surviving cell fraction was determined by an MTS assay, and erlotinib IC 50 values were determined for each condition (** P

    Article Snippet: Western blot analysis Cell extracts were analyzed by Western blotting with primary antibodies against RHOB, ERK1/2 (Santa Cruz Biotechnology), p‐ERK (T202/Y204), p‐AKT (S473), AKT, pGSK3 (S9), GSK3, p‐EGFR (T1173), EGFR, PARP, cleaved caspase‐3, caspase‐3 (Cell Signaling Technology), or actin (Chemicon).

    Techniques: Inhibition, Expressing, Transduction, Western Blot, Over Expression, MTS Assay

    Kaplan Meier curves of disease free survival of risk groups for TNBC patients. The patients were stratified into different risk groups. Two low basal (EGFR≤15% and CK5/6≤50%) risk groups: group 1 (n = 20) with low expressions/values of EGFR (≤15%), CK5/6 (≤50%), Ki-67 (≤50%), pT (≤ 3), and pN (≤ 1), and group 2 (n = 30) with low expressions of EGFR and CK5/6, and any high expressions/values of Ki-67, pT, and pN. Two high basal risk groups: group 3 (n = 106) with single high basal expression (EGFR > 15% or CK5/6 > 50%), and group 4 (n = 36) with double high basal expressions (EGFR > 15% and CK5/6 > 50%).

    Journal: PLoS ONE

    Article Title: Stratification of Prognosis of Triple-Negative Breast Cancer Patients Using Combinatorial Biomarkers

    doi: 10.1371/journal.pone.0149661

    Figure Lengend Snippet: Kaplan Meier curves of disease free survival of risk groups for TNBC patients. The patients were stratified into different risk groups. Two low basal (EGFR≤15% and CK5/6≤50%) risk groups: group 1 (n = 20) with low expressions/values of EGFR (≤15%), CK5/6 (≤50%), Ki-67 (≤50%), pT (≤ 3), and pN (≤ 1), and group 2 (n = 30) with low expressions of EGFR and CK5/6, and any high expressions/values of Ki-67, pT, and pN. Two high basal risk groups: group 3 (n = 106) with single high basal expression (EGFR > 15% or CK5/6 > 50%), and group 4 (n = 36) with double high basal expressions (EGFR > 15% and CK5/6 > 50%).

    Article Snippet: Immunostaining for CK 5/6, and EGFR was performed using monoclonal antibodies D5/16 B4 for CK5/6 (Dako, Glostrup, Denmark); and 2-18C9 for EGFR (Dako, Glostrup, Denmark).

    Techniques: Expressing

    Immunoreactivity of 69-yr-old patient with T1bN0M0 TNBC. (A) Hematoxylin and Eosin (H E), (B) EGFR ( > 70%), (C) CK5/6 ( > 70%), (D) ER, (E) PR, (F) HER2, (G) Ki-67 ( > 60%), and (H) P53 ( > 90%). The patient was undergone BCT, had 3.8 event free survival and 22.8 months overall survival.

    Journal: PLoS ONE

    Article Title: Stratification of Prognosis of Triple-Negative Breast Cancer Patients Using Combinatorial Biomarkers

    doi: 10.1371/journal.pone.0149661

    Figure Lengend Snippet: Immunoreactivity of 69-yr-old patient with T1bN0M0 TNBC. (A) Hematoxylin and Eosin (H E), (B) EGFR ( > 70%), (C) CK5/6 ( > 70%), (D) ER, (E) PR, (F) HER2, (G) Ki-67 ( > 60%), and (H) P53 ( > 90%). The patient was undergone BCT, had 3.8 event free survival and 22.8 months overall survival.

    Article Snippet: Immunostaining for CK 5/6, and EGFR was performed using monoclonal antibodies D5/16 B4 for CK5/6 (Dako, Glostrup, Denmark); and 2-18C9 for EGFR (Dako, Glostrup, Denmark).

    Techniques:

    Kaplan Meier curves of disease free survival for basal biomarkers. (a) EGFR at cut-off level 15% with log-rank p = 0.0016, (b) time-dependent ROC analysis of EGFR with AUC = 0.723, where cutoff point 15% (red circled), (c) CK5/6 at cut-off level 50% with log-rank p = 0.0066, and (d) the significance of survival difference at different cutoff values for EGFR and CK5/6. The most significant cutoff values were red-circled with 15% for EGFR and 50% for CK5/6.

    Journal: PLoS ONE

    Article Title: Stratification of Prognosis of Triple-Negative Breast Cancer Patients Using Combinatorial Biomarkers

    doi: 10.1371/journal.pone.0149661

    Figure Lengend Snippet: Kaplan Meier curves of disease free survival for basal biomarkers. (a) EGFR at cut-off level 15% with log-rank p = 0.0016, (b) time-dependent ROC analysis of EGFR with AUC = 0.723, where cutoff point 15% (red circled), (c) CK5/6 at cut-off level 50% with log-rank p = 0.0066, and (d) the significance of survival difference at different cutoff values for EGFR and CK5/6. The most significant cutoff values were red-circled with 15% for EGFR and 50% for CK5/6.

    Article Snippet: Immunostaining for CK 5/6, and EGFR was performed using monoclonal antibodies D5/16 B4 for CK5/6 (Dako, Glostrup, Denmark); and 2-18C9 for EGFR (Dako, Glostrup, Denmark).

    Techniques:

    Comparing the standard EGFR PharmDx™ with the modified EGFR PharmDx™ and the highsensitivity EGFR immunostaining

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: High-sensitivity epidermal growth factor receptor immunostaining for colorectal carcinomas, compared with EGFR PharmDx(TM): A study of diagnostic accuracy

    doi:

    Figure Lengend Snippet: Comparing the standard EGFR PharmDx™ with the modified EGFR PharmDx™ and the highsensitivity EGFR immunostaining

    Article Snippet: Buckley AF, Kakar S. Comparison of the Dako EGFR PharmDx kit and Zymed EGFR antibody for assessment of EGFR status in colorectal adenocarcinoma.

    Techniques: Modification, Immunostaining

    Comparative study of two colon cancer cases using the standard and modified EGFR PharmDx™ and high-sensitivity EGFR immunostaining with DAK-H1-WT and Novolink. (A, D, G) standard EGFR PharmDx™, (B, E, H) modified EGFR PharmDx™

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: High-sensitivity epidermal growth factor receptor immunostaining for colorectal carcinomas, compared with EGFR PharmDx(TM): A study of diagnostic accuracy

    doi:

    Figure Lengend Snippet: Comparative study of two colon cancer cases using the standard and modified EGFR PharmDx™ and high-sensitivity EGFR immunostaining with DAK-H1-WT and Novolink. (A, D, G) standard EGFR PharmDx™, (B, E, H) modified EGFR PharmDx™

    Article Snippet: Buckley AF, Kakar S. Comparison of the Dako EGFR PharmDx kit and Zymed EGFR antibody for assessment of EGFR status in colorectal adenocarcinoma.

    Techniques: Modification, Immunostaining

    LMP1 increased the binding ability of transcription factors EGFR and STAT3 to cyclin D1 promoter in vitro . (A) STAT3 binding activities within the cyclin D1 promoter were examined by EMSA. A biotin-labeled wild-type STAT3 oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type STAT3 (lane 4), unlabeled mutant STAT3 oligonucleotides (lane 5), or noncompetitive unlabeled NF-κB oligonucleotide (NS, lane 6). Biotin-labeled mutant STAT3 oligonucleotide probe was incubated with nuclear extracts of the indicated NPC cell lines (lanes 8–9). (B) Ten micrograms of nuclear extracts were pre-incubated with biotin-labeled STAT3 oligonucleotide probe in the presence of inhibitors directed against different phosphorylation sites of STAT3 (indicated above each lane). (C) The biotin-labeled wild-type EGFR oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type EGFR (lane 4), unlabeled mutant EGFR oligonucleotides (lane 6) or noncompetitive unlabeled NFκB oligonucleotide (NS, lane 7), and then EGFR DNA binding activities were examined by EMSA. (D-E) The nuclear extracts of CNE1 and CNE1-LMP1 cells were pre-incubated with biotin-labeled EGFR oligonucleotide probe in the presence of inhibitors AG1478, directed against phosphorylation of EGFR, or DNAzyme 1 (DZ1), targeting LMP1. RD: relative density.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Epstein-Barr Virus encoded LMP1 regulates cyclin D1 promoter activity by nuclear EGFR and STAT3 in CNE1 cells

    doi: 10.1186/1756-9966-32-90

    Figure Lengend Snippet: LMP1 increased the binding ability of transcription factors EGFR and STAT3 to cyclin D1 promoter in vitro . (A) STAT3 binding activities within the cyclin D1 promoter were examined by EMSA. A biotin-labeled wild-type STAT3 oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type STAT3 (lane 4), unlabeled mutant STAT3 oligonucleotides (lane 5), or noncompetitive unlabeled NF-κB oligonucleotide (NS, lane 6). Biotin-labeled mutant STAT3 oligonucleotide probe was incubated with nuclear extracts of the indicated NPC cell lines (lanes 8–9). (B) Ten micrograms of nuclear extracts were pre-incubated with biotin-labeled STAT3 oligonucleotide probe in the presence of inhibitors directed against different phosphorylation sites of STAT3 (indicated above each lane). (C) The biotin-labeled wild-type EGFR oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type EGFR (lane 4), unlabeled mutant EGFR oligonucleotides (lane 6) or noncompetitive unlabeled NFκB oligonucleotide (NS, lane 7), and then EGFR DNA binding activities were examined by EMSA. (D-E) The nuclear extracts of CNE1 and CNE1-LMP1 cells were pre-incubated with biotin-labeled EGFR oligonucleotide probe in the presence of inhibitors AG1478, directed against phosphorylation of EGFR, or DNAzyme 1 (DZ1), targeting LMP1. RD: relative density.

    Article Snippet: The antibodies used were as follows: EGFR (sc-03-G, Santa Cruz, U.S.A.), p-EGFR (sc-12351, Santa Cruz, U.S.A.), STAT3 (#9132, Cell Signaling Technology, U.S.A.), p-STAT3 (#9131, Cell Signaling Technology, U.S.A.), β-actin (sc-8432, Santa Cruz, U.S.A.), α-tubulin (sc-5286, Santa Cruz, U.S.A.), Nucleolin (sc-8031, Santa Cruz, U.S.A.), cyclin D1 (Cat# 2261–1, Epitomics, U.S.A.).

    Techniques: Binding Assay, In Vitro, Labeling, Incubation, Mutagenesis

    LMP1 induced co-localization of EGFR and STAT3 in the nucleus. Endogenous association of EGFR (A) with STAT3 (B) in NPC cells without or with LMP1 expression. Equal amounts of fractionated cellular proteins were immunoprecipitated with an anti-EGFR or anti-STAT3 antibody and loaded for Western blotting. Input samples from equal amounts of proteins blotted for EGFR, STAT3, nucleolin, and α-tubulin are shown as loading and fractionation controls. N: nuclear fraction, C: cytosolic fraction, IB: immunoblot.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Epstein-Barr Virus encoded LMP1 regulates cyclin D1 promoter activity by nuclear EGFR and STAT3 in CNE1 cells

    doi: 10.1186/1756-9966-32-90

    Figure Lengend Snippet: LMP1 induced co-localization of EGFR and STAT3 in the nucleus. Endogenous association of EGFR (A) with STAT3 (B) in NPC cells without or with LMP1 expression. Equal amounts of fractionated cellular proteins were immunoprecipitated with an anti-EGFR or anti-STAT3 antibody and loaded for Western blotting. Input samples from equal amounts of proteins blotted for EGFR, STAT3, nucleolin, and α-tubulin are shown as loading and fractionation controls. N: nuclear fraction, C: cytosolic fraction, IB: immunoblot.

    Article Snippet: The antibodies used were as follows: EGFR (sc-03-G, Santa Cruz, U.S.A.), p-EGFR (sc-12351, Santa Cruz, U.S.A.), STAT3 (#9132, Cell Signaling Technology, U.S.A.), p-STAT3 (#9131, Cell Signaling Technology, U.S.A.), β-actin (sc-8432, Santa Cruz, U.S.A.), α-tubulin (sc-5286, Santa Cruz, U.S.A.), Nucleolin (sc-8031, Santa Cruz, U.S.A.), cyclin D1 (Cat# 2261–1, Epitomics, U.S.A.).

    Techniques: Expressing, Immunoprecipitation, Western Blot, Fractionation

    Identification of an EGFR and STAT3 response element in the cyclin D1 promoter. (A) Schematic diagram of mutant cyclin D1 promoter constructs are shown. The expansion for EGFR and STAT3 binding site illustrates the wild-type sequence and frames the nucleotides replaced by mutations. (B-C) Dual luciferase-reporter assays were performed in LMP1-negative and LMP-positive CNE1 cells after co-transfection of a wild type or mutant cyclin D1 promoter-reporter construct, plasmids expressing wild-type EGFR or STAT3, and a Renilla luciferase transfection control plasmid. The fold induction by EGFR and STAT3 is displayed as the ratio of promoter activity obtained with wild-type compared to the DNA-binding mutant. (mean ± SD, n = 3, * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Epstein-Barr Virus encoded LMP1 regulates cyclin D1 promoter activity by nuclear EGFR and STAT3 in CNE1 cells

    doi: 10.1186/1756-9966-32-90

    Figure Lengend Snippet: Identification of an EGFR and STAT3 response element in the cyclin D1 promoter. (A) Schematic diagram of mutant cyclin D1 promoter constructs are shown. The expansion for EGFR and STAT3 binding site illustrates the wild-type sequence and frames the nucleotides replaced by mutations. (B-C) Dual luciferase-reporter assays were performed in LMP1-negative and LMP-positive CNE1 cells after co-transfection of a wild type or mutant cyclin D1 promoter-reporter construct, plasmids expressing wild-type EGFR or STAT3, and a Renilla luciferase transfection control plasmid. The fold induction by EGFR and STAT3 is displayed as the ratio of promoter activity obtained with wild-type compared to the DNA-binding mutant. (mean ± SD, n = 3, * p

    Article Snippet: The antibodies used were as follows: EGFR (sc-03-G, Santa Cruz, U.S.A.), p-EGFR (sc-12351, Santa Cruz, U.S.A.), STAT3 (#9132, Cell Signaling Technology, U.S.A.), p-STAT3 (#9131, Cell Signaling Technology, U.S.A.), β-actin (sc-8432, Santa Cruz, U.S.A.), α-tubulin (sc-5286, Santa Cruz, U.S.A.), Nucleolin (sc-8031, Santa Cruz, U.S.A.), cyclin D1 (Cat# 2261–1, Epitomics, U.S.A.).

    Techniques: Mutagenesis, Construct, Binding Assay, Sequencing, Luciferase, Cotransfection, Expressing, Transfection, Plasmid Preparation, Activity Assay

    Inhibitors and dominant negative mutants targeting the EGFR and STAT3 pathways attenuated LMP1-augmented cyclin D1 promoter activity. (A-B) Stable expression of EGFR-DN and STAT3β inhibited the LMP1-increased activity of cyclin D1. The indicated NPC cell lines were transfected with a cyclin D1 promoter-reporter construct, a Renilla luciferase transfection control plasmid, and an EGFR-DN or STAT3-β expression plasmid. Twenty-four hrs. after transfection, the cells were treated with DNAzymes or a control oligo (2 μM) for 12 hrs. Cells were harvested at 36 hrs. after transfection and subjected to the luciferase assay. Firefly luciferase was measured and normalized to Renilla luciferase activity. The results were expressed as fold induction of the reporter activity in vector-transfected CNE1 cells, which was assigned a value of 1. (mean ± SD, n =3, * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Epstein-Barr Virus encoded LMP1 regulates cyclin D1 promoter activity by nuclear EGFR and STAT3 in CNE1 cells

    doi: 10.1186/1756-9966-32-90

    Figure Lengend Snippet: Inhibitors and dominant negative mutants targeting the EGFR and STAT3 pathways attenuated LMP1-augmented cyclin D1 promoter activity. (A-B) Stable expression of EGFR-DN and STAT3β inhibited the LMP1-increased activity of cyclin D1. The indicated NPC cell lines were transfected with a cyclin D1 promoter-reporter construct, a Renilla luciferase transfection control plasmid, and an EGFR-DN or STAT3-β expression plasmid. Twenty-four hrs. after transfection, the cells were treated with DNAzymes or a control oligo (2 μM) for 12 hrs. Cells were harvested at 36 hrs. after transfection and subjected to the luciferase assay. Firefly luciferase was measured and normalized to Renilla luciferase activity. The results were expressed as fold induction of the reporter activity in vector-transfected CNE1 cells, which was assigned a value of 1. (mean ± SD, n =3, * p

    Article Snippet: The antibodies used were as follows: EGFR (sc-03-G, Santa Cruz, U.S.A.), p-EGFR (sc-12351, Santa Cruz, U.S.A.), STAT3 (#9132, Cell Signaling Technology, U.S.A.), p-STAT3 (#9131, Cell Signaling Technology, U.S.A.), β-actin (sc-8432, Santa Cruz, U.S.A.), α-tubulin (sc-5286, Santa Cruz, U.S.A.), Nucleolin (sc-8031, Santa Cruz, U.S.A.), cyclin D1 (Cat# 2261–1, Epitomics, U.S.A.).

    Techniques: Dominant Negative Mutation, Activity Assay, Expressing, Transfection, Construct, Luciferase, Plasmid Preparation

    Cyclin D1 expression is reduced in CNE1-LMP1 cells after treatment with EGFR siRNA and STAT3 siRNA. (A) Dual luciferase-reporter assays were performed in CNE1-LMP1 cells after co-transfection with either control siRNA (siControl), EGFR siRNA (siEGFR), or STAT3 siRNA (siSTAT3) in addition to cyclin D1 promoter-reporter constructs and a Renilla luciferase transfection control plasmid. Firefly luciferase was measured and normalized to Renilla luciferase activity. The fold change in cyclin D1 expression by the indicated siRNA is displayed in each case. The control siRNA served as a non-targeting control. (mean ± SD, n =3, * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Epstein-Barr Virus encoded LMP1 regulates cyclin D1 promoter activity by nuclear EGFR and STAT3 in CNE1 cells

    doi: 10.1186/1756-9966-32-90

    Figure Lengend Snippet: Cyclin D1 expression is reduced in CNE1-LMP1 cells after treatment with EGFR siRNA and STAT3 siRNA. (A) Dual luciferase-reporter assays were performed in CNE1-LMP1 cells after co-transfection with either control siRNA (siControl), EGFR siRNA (siEGFR), or STAT3 siRNA (siSTAT3) in addition to cyclin D1 promoter-reporter constructs and a Renilla luciferase transfection control plasmid. Firefly luciferase was measured and normalized to Renilla luciferase activity. The fold change in cyclin D1 expression by the indicated siRNA is displayed in each case. The control siRNA served as a non-targeting control. (mean ± SD, n =3, * p

    Article Snippet: The antibodies used were as follows: EGFR (sc-03-G, Santa Cruz, U.S.A.), p-EGFR (sc-12351, Santa Cruz, U.S.A.), STAT3 (#9132, Cell Signaling Technology, U.S.A.), p-STAT3 (#9131, Cell Signaling Technology, U.S.A.), β-actin (sc-8432, Santa Cruz, U.S.A.), α-tubulin (sc-5286, Santa Cruz, U.S.A.), Nucleolin (sc-8031, Santa Cruz, U.S.A.), cyclin D1 (Cat# 2261–1, Epitomics, U.S.A.).

    Techniques: Expressing, Luciferase, Cotransfection, Construct, Transfection, Plasmid Preparation, Activity Assay

    LMP1 affected the interaction of EGFR and STAT3. Two mg of protein from cell lysates were immunoprecipitated with an anti-EGFR antibody (A) or anti-STAT3 antibody (B) and analyzed by Western blotting with a STAT3 and EGFR antibodies. Negative controls included immunoprecipitation with an unrelated antibody (IgG). ®-actin were used as an internal control of Inuput. The bottom panels show the 50 μg of input materials. IP: immunoprecipitation, IB: immunoblot, kDa: kilodalton.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Epstein-Barr Virus encoded LMP1 regulates cyclin D1 promoter activity by nuclear EGFR and STAT3 in CNE1 cells

    doi: 10.1186/1756-9966-32-90

    Figure Lengend Snippet: LMP1 affected the interaction of EGFR and STAT3. Two mg of protein from cell lysates were immunoprecipitated with an anti-EGFR antibody (A) or anti-STAT3 antibody (B) and analyzed by Western blotting with a STAT3 and EGFR antibodies. Negative controls included immunoprecipitation with an unrelated antibody (IgG). ®-actin were used as an internal control of Inuput. The bottom panels show the 50 μg of input materials. IP: immunoprecipitation, IB: immunoblot, kDa: kilodalton.

    Article Snippet: The antibodies used were as follows: EGFR (sc-03-G, Santa Cruz, U.S.A.), p-EGFR (sc-12351, Santa Cruz, U.S.A.), STAT3 (#9132, Cell Signaling Technology, U.S.A.), p-STAT3 (#9131, Cell Signaling Technology, U.S.A.), β-actin (sc-8432, Santa Cruz, U.S.A.), α-tubulin (sc-5286, Santa Cruz, U.S.A.), Nucleolin (sc-8031, Santa Cruz, U.S.A.), cyclin D1 (Cat# 2261–1, Epitomics, U.S.A.).

    Techniques: Immunoprecipitation, Western Blot

    Resistance to Th1 cytokine/trastuzumab-mediated class I restoration and HER2 369-377 -CD8 + T-cell targeting of HER2-expressing cancers by EGF/Heregulin is rescued with inhibition of EGFR and HER3 signaling (A) Effect of EGFR- and HER3-mediated signaling on class I restoration by trastuzumab and Th1 cytokines. Trastuzumab-treated HER2 high BT-474 cells were serum starved and activated with EGF, Heregulin, or both, followed by IFNγ and TNFα treatment. Harvested cells were assessed for HLA-ABC expression by flow cytometry. Results are representative of three experiments, and expressed as mean HLA-ABC MCF ± SEM. (B) Trastuzumab/IFNγ/TNFα-treated HER2 high SK-BR-3 ( left panel ) and BT-474 ( right panel ) cells with or without EGF + Heregulin activation, were subsequently treated with anti-EGFR + anti-HER3 neutralizing or IgG1 isotype control antibodies. Harvested cells were assessed for HLA-ABC expression by flow cytometry. In representative panels, filled traces represent isotype-matched control staining, and open traces represent HLA-ABC staining. Color-coded cell treatments are indicated in the legend. Adjoining results in histograms are representative of three experiments, and expressed as mean HLA-ABC MCF ± SEM; cell treatments are indicated below the histograms. (C) Following treatments indicated in (B) above, CFSE-labeled HER2 high SK-BR-3 cells were co-cultured 1:1 with HER2 369-377 -sensitized CD8 + T cells. Tumor cells were harvested, stained with 7-AAD and FITC:anti-CD8, and CSFE + 7-AAD + CD8 - cells (apoptotic) were assessed by flow cytometry. Results are representative of three experiments, and expressed as % apoptotic tumor cells±SEM ( % lysis ) in co-culture minus background. *p≤0.05, **p

    Journal: Cancer immunology research

    Article Title: CD4+ T-helper Type 1 Cytokines and Trastuzumab Facilitate CD8+ T-cell Targeting of HER-2/neu-expressing Cancers

    doi: 10.1158/2326-6066.CIR-14-0208

    Figure Lengend Snippet: Resistance to Th1 cytokine/trastuzumab-mediated class I restoration and HER2 369-377 -CD8 + T-cell targeting of HER2-expressing cancers by EGF/Heregulin is rescued with inhibition of EGFR and HER3 signaling (A) Effect of EGFR- and HER3-mediated signaling on class I restoration by trastuzumab and Th1 cytokines. Trastuzumab-treated HER2 high BT-474 cells were serum starved and activated with EGF, Heregulin, or both, followed by IFNγ and TNFα treatment. Harvested cells were assessed for HLA-ABC expression by flow cytometry. Results are representative of three experiments, and expressed as mean HLA-ABC MCF ± SEM. (B) Trastuzumab/IFNγ/TNFα-treated HER2 high SK-BR-3 ( left panel ) and BT-474 ( right panel ) cells with or without EGF + Heregulin activation, were subsequently treated with anti-EGFR + anti-HER3 neutralizing or IgG1 isotype control antibodies. Harvested cells were assessed for HLA-ABC expression by flow cytometry. In representative panels, filled traces represent isotype-matched control staining, and open traces represent HLA-ABC staining. Color-coded cell treatments are indicated in the legend. Adjoining results in histograms are representative of three experiments, and expressed as mean HLA-ABC MCF ± SEM; cell treatments are indicated below the histograms. (C) Following treatments indicated in (B) above, CFSE-labeled HER2 high SK-BR-3 cells were co-cultured 1:1 with HER2 369-377 -sensitized CD8 + T cells. Tumor cells were harvested, stained with 7-AAD and FITC:anti-CD8, and CSFE + 7-AAD + CD8 - cells (apoptotic) were assessed by flow cytometry. Results are representative of three experiments, and expressed as % apoptotic tumor cells±SEM ( % lysis ) in co-culture minus background. *p≤0.05, **p

    Article Snippet: HER2-expressing cells were treated with the following, either alone or in designated combinations: rhTNFα, rhIFNγ (BD Biosciences), trastuzumab (Genentech), lapatinib (Santa Cruz Biotechnology); rh-EGF (BD Biosciences), rh-Heregulin (Sigma-Aldrich); neutralizing anti-EGFR (LA1) and/or anti-HER3 (H3.105.5) antibodies, or IgG1 isotype control antibody (all Millipore); and neutralizing anti-PD-1 (MIH1 ( )) or IgG1 isotype control (eBiosciences).

    Techniques: Expressing, Inhibition, Flow Cytometry, Cytometry, Activation Assay, Staining, Labeling, Cell Culture, Lysis, Co-Culture Assay

    GGA2 colocalizes and interacts with EGFR in vivo . ( a ) ARPE-19 cells overexpressing GFP-tagged GGA2 were fixed and double immunofluorescence staining was performed using antibodies against EGFR (red) and TGN46, EEA1, or cathepsin D (blue). Arrows and arrowheads indicate colocalization of three signals in the TGN and peripheral structures, respectively. Bar, 20 μm. ( b ) ARPE-19 cells were fixed for triple immunofluorescence analyses using antibodies against Alexa488-EEA1 (green), endogenous GGA2 (red) and EGFR (blue). Arrowheads indicate colocalization of three signals. Bar, 20 μm. ( c ) ARPE-19 cells transfected with siCtrl, siGGA2, or siEGFR were processed for PLA to detect GGA2-EGFR or GGA2-CIMPR interaction (green). Nuclei were stained with DAPI (blue). Bars, 20 μm.

    Journal: Scientific Reports

    Article Title: GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth

    doi: 10.1038/s41598-018-19542-4

    Figure Lengend Snippet: GGA2 colocalizes and interacts with EGFR in vivo . ( a ) ARPE-19 cells overexpressing GFP-tagged GGA2 were fixed and double immunofluorescence staining was performed using antibodies against EGFR (red) and TGN46, EEA1, or cathepsin D (blue). Arrows and arrowheads indicate colocalization of three signals in the TGN and peripheral structures, respectively. Bar, 20 μm. ( b ) ARPE-19 cells were fixed for triple immunofluorescence analyses using antibodies against Alexa488-EEA1 (green), endogenous GGA2 (red) and EGFR (blue). Arrowheads indicate colocalization of three signals. Bar, 20 μm. ( c ) ARPE-19 cells transfected with siCtrl, siGGA2, or siEGFR were processed for PLA to detect GGA2-EGFR or GGA2-CIMPR interaction (green). Nuclei were stained with DAPI (blue). Bars, 20 μm.

    Article Snippet: For quantification of EGFR signal in GGA2-depleted ARPE-19 cells re-expressing HA-GGA2kd-resi , fixed cells were immunostained with anti-EGFR antibody (Millipore) without permeabilization, and then immunostained with anti-HA antibody following permeabilization.

    Techniques: In Vivo, Double Immunofluorescence Staining, Immunofluorescence, Transfection, Proximity Ligation Assay, Staining

    GGA2-depletion facilitates lysosomal degradation of EGFR via post-Golgi compartments. ( a ) Control (siCtrl) and GGA2-depleted (siGGA2) ARPE-19 cells were pulse-labeled with [ 35 . ( b ) Signals for EGFR were quantified in three experiments and were plotted as means ± SD. Differences between siCtrl and siGGA2 at each time point were identified using Student’s t -test; * P

    Journal: Scientific Reports

    Article Title: GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth

    doi: 10.1038/s41598-018-19542-4

    Figure Lengend Snippet: GGA2-depletion facilitates lysosomal degradation of EGFR via post-Golgi compartments. ( a ) Control (siCtrl) and GGA2-depleted (siGGA2) ARPE-19 cells were pulse-labeled with [ 35 . ( b ) Signals for EGFR were quantified in three experiments and were plotted as means ± SD. Differences between siCtrl and siGGA2 at each time point were identified using Student’s t -test; * P

    Article Snippet: For quantification of EGFR signal in GGA2-depleted ARPE-19 cells re-expressing HA-GGA2kd-resi , fixed cells were immunostained with anti-EGFR antibody (Millipore) without permeabilization, and then immunostained with anti-HA antibody following permeabilization.

    Techniques: Labeling

    GGA2 VHS-GAT recognizes EGFR juxtamembrane (jxt) region. ( a ) Domain structures of EGFR and GGA2 mutants used in the binding assay are shown. Numbers indicate amino acid sequences of the proteins. N108A mutation site is indicated in VHS-GAT. TMD, transmembrane domain. ( b and c ) HEK293 cells were transfected with GFP tagged with entire cytoplasmic domain of EGFR (cyto), or with its jxt, kinase, or tail region. Cell lysates were mixed with glutathione sepharose and GST-GGA2 VHS-GAT (GST-VG[WT]) or its N108A mutant (GST-VG[NA]). Bound proteins were then eluted and analyzed using SDS-PAGE and immunoblotting with anti-GFP or -CIMPR (only for [b]) antibody as indicated (Ab). ( d .

    Journal: Scientific Reports

    Article Title: GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth

    doi: 10.1038/s41598-018-19542-4

    Figure Lengend Snippet: GGA2 VHS-GAT recognizes EGFR juxtamembrane (jxt) region. ( a ) Domain structures of EGFR and GGA2 mutants used in the binding assay are shown. Numbers indicate amino acid sequences of the proteins. N108A mutation site is indicated in VHS-GAT. TMD, transmembrane domain. ( b and c ) HEK293 cells were transfected with GFP tagged with entire cytoplasmic domain of EGFR (cyto), or with its jxt, kinase, or tail region. Cell lysates were mixed with glutathione sepharose and GST-GGA2 VHS-GAT (GST-VG[WT]) or its N108A mutant (GST-VG[NA]). Bound proteins were then eluted and analyzed using SDS-PAGE and immunoblotting with anti-GFP or -CIMPR (only for [b]) antibody as indicated (Ab). ( d .

    Article Snippet: For quantification of EGFR signal in GGA2-depleted ARPE-19 cells re-expressing HA-GGA2kd-resi , fixed cells were immunostained with anti-EGFR antibody (Millipore) without permeabilization, and then immunostained with anti-HA antibody following permeabilization.

    Techniques: Binding Assay, Mutagenesis, Transfection, SDS Page

    Simultaneous depletion of GGA1 or GGA3 with GGA2 restores EGFR expression. ( a and b ) ARPE-19 cells were transfected with control (siCtrl), GGA1 (siGGA1), GGA2 (siGGA2), or GGA3 (siGGA3) siRNAs alone, or with a combination of siGGA2 and siGGA1, or siGGA2 and siGGA3 as indicated. Cells were then fixed for immunofluorescence staining with anti-EGFR antibody ( a ), or were lysed for western blot analysis with antibodies against EGFR, GGA1, GGA2, GGA3, and GAPDH ( b ). Nuclei were labeled with Hoechst 33342. Bar, 20 μm. ( c . ( d ) ARPE-19 cells transfected with siCtrl, siGGA1, or siGGA3 were used for PLA to detect GGA2-EGFR interaction (green). Nuclei were stained with DAPI (blue). Bar, 20 μm. ( e ) PLA signals/cell were counted and mean values ± SD (n = 575 [siCtrl], n = 482 [siGGA1], and n = 448 [siGGA3]) were plotted. Differences were analyzed by one-way ANOVA ( P

    Journal: Scientific Reports

    Article Title: GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth

    doi: 10.1038/s41598-018-19542-4

    Figure Lengend Snippet: Simultaneous depletion of GGA1 or GGA3 with GGA2 restores EGFR expression. ( a and b ) ARPE-19 cells were transfected with control (siCtrl), GGA1 (siGGA1), GGA2 (siGGA2), or GGA3 (siGGA3) siRNAs alone, or with a combination of siGGA2 and siGGA1, or siGGA2 and siGGA3 as indicated. Cells were then fixed for immunofluorescence staining with anti-EGFR antibody ( a ), or were lysed for western blot analysis with antibodies against EGFR, GGA1, GGA2, GGA3, and GAPDH ( b ). Nuclei were labeled with Hoechst 33342. Bar, 20 μm. ( c . ( d ) ARPE-19 cells transfected with siCtrl, siGGA1, or siGGA3 were used for PLA to detect GGA2-EGFR interaction (green). Nuclei were stained with DAPI (blue). Bar, 20 μm. ( e ) PLA signals/cell were counted and mean values ± SD (n = 575 [siCtrl], n = 482 [siGGA1], and n = 448 [siGGA3]) were plotted. Differences were analyzed by one-way ANOVA ( P

    Article Snippet: For quantification of EGFR signal in GGA2-depleted ARPE-19 cells re-expressing HA-GGA2kd-resi , fixed cells were immunostained with anti-EGFR antibody (Millipore) without permeabilization, and then immunostained with anti-HA antibody following permeabilization.

    Techniques: Expressing, Transfection, Immunofluorescence, Staining, Western Blot, Labeling, Proximity Ligation Assay

    GGA2-depletion causes a drastic decrease in the EGFR protein expression. ( a . ( b ) Band intensities for EGFR were quantified in three or four experiments and mean values ± standard deviations (SD) were plotted. Differences between each GGA siRNA and siCtrl were analyzed by one-way ANOVA ( P

    Journal: Scientific Reports

    Article Title: GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth

    doi: 10.1038/s41598-018-19542-4

    Figure Lengend Snippet: GGA2-depletion causes a drastic decrease in the EGFR protein expression. ( a . ( b ) Band intensities for EGFR were quantified in three or four experiments and mean values ± standard deviations (SD) were plotted. Differences between each GGA siRNA and siCtrl were analyzed by one-way ANOVA ( P

    Article Snippet: For quantification of EGFR signal in GGA2-depleted ARPE-19 cells re-expressing HA-GGA2kd-resi , fixed cells were immunostained with anti-EGFR antibody (Millipore) without permeabilization, and then immunostained with anti-HA antibody following permeabilization.

    Techniques: Expressing

    EGF-induced EGFR signaling and cell growth are impaired in GGA2-depleted cells. ( a . ( b ) Increase in the ratio of P-MAPK to MAPK after EGF stimulation was calculated and normalized to the value of siCtrl. Mean (±SD) of three experiments were plotted. Statistical difference between the control and each GGA2 siRNA was identified using Student’s t -test (** P

    Journal: Scientific Reports

    Article Title: GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth

    doi: 10.1038/s41598-018-19542-4

    Figure Lengend Snippet: EGF-induced EGFR signaling and cell growth are impaired in GGA2-depleted cells. ( a . ( b ) Increase in the ratio of P-MAPK to MAPK after EGF stimulation was calculated and normalized to the value of siCtrl. Mean (±SD) of three experiments were plotted. Statistical difference between the control and each GGA2 siRNA was identified using Student’s t -test (** P

    Article Snippet: For quantification of EGFR signal in GGA2-depleted ARPE-19 cells re-expressing HA-GGA2kd-resi , fixed cells were immunostained with anti-EGFR antibody (Millipore) without permeabilization, and then immunostained with anti-HA antibody following permeabilization.

    Techniques:

    Downregulation of EGFR and p1068EGFR expression by EGCG and IIF treatments in BE(2)-C neuroblastoma cells. Cells were treated with 20 μg/mL EGCG and 10 μM IIF, individually and in combination for 24 h. ( A ) Proteins (50 μg) from total cell lysate were subjected to SDS–PAGE and Western blot analysis of EGFR and p1068EGFR expression after 24 h treatments. Actin was used as a loading control. RT-PCR analysis of EGFR ( B ) and NDRG1 ( C ) in control and treated cells. β-actin was used as a control. The values were normalized to the untreated controls. The results are expressed as the average ± SE of three independent experiments. * p

    Journal: Nutrients

    Article Title: Epigallocatechin-3-gallate and 6-OH-11-O-Hydroxyphenanthrene Limit BE(2)-C Neuroblastoma Cell Growth and Neurosphere Formation In Vitro

    doi: 10.3390/nu10091141

    Figure Lengend Snippet: Downregulation of EGFR and p1068EGFR expression by EGCG and IIF treatments in BE(2)-C neuroblastoma cells. Cells were treated with 20 μg/mL EGCG and 10 μM IIF, individually and in combination for 24 h. ( A ) Proteins (50 μg) from total cell lysate were subjected to SDS–PAGE and Western blot analysis of EGFR and p1068EGFR expression after 24 h treatments. Actin was used as a loading control. RT-PCR analysis of EGFR ( B ) and NDRG1 ( C ) in control and treated cells. β-actin was used as a control. The values were normalized to the untreated controls. The results are expressed as the average ± SE of three independent experiments. * p

    Article Snippet: Antibodies: anti-RARα and anti-RXRγ (Tema Ricerca, Bologna, Italy), anti-EGFR (Thermo Scientific, Waltham, MA, USA), anti-p1068EGFR (Novex, Life Technologies, Carlsbad, CA, USA), anti-Bcl-2 (Sigma-Aldrich, St. Louis, MO, USA), anti-Bax (Applied Biosystem, Monza, Italy) anti-PARP (Santa Cruz Biotechnology, Dallas, TX, USA), anti-COX-2 (Sigma-Aldrich, St. Louis, MO, USA), anti-N-MYC, anti MMP-2, MMP-9, and anti-TIMP-1 (all from Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-tubulin (Sigma-Aldrich, St. Louis, MO, USA), anti-rabbit, and anti-mouse peroxidase conjugated antibodies (GE Healthcare, Milan, Italy).

    Techniques: Expressing, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction