egfr  (Thermo Fisher)


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    Name:
    KDR VEGFR2 Recombinant Human Protein
    Description:
    Kinase insert domain receptor KDR recombinant human protein is supplied as a lyophilized powder It is suitable for use in analysis of protein structure and performing cell based assays It can also be used as an immunogen as a protein standard or in other research applications This recombinant protein was expressed from a DNA sequence encoding the extracellular domain of human KDR NP 002244 1 Met 1 Glu 764 fused with a polyhistidine tag at the C terminus Activity Using the Octet RED System The affinity constant Kd of human KDR His bound to biotinylated human NRP2 VEGF165 was 5 9 nM Formulation Lyophilized in 140 mM NaCl 2 7 mM KCl 10 mM Na2HPO4 1 8 mM KH2PO4 pH 7 4 5 Mannitol 5 Trehalose 0 02 Tween 80 Reconstitution Dissolve the protein in sterile double distilled water to a concentration of 0 2 mg ml or lower It is recommended that the protein be aliquoted and be used as soon as possible Store aliquots under sterile conditions at 20°C Avoid repeated freeze thaw cycles Expiration Date Expires one year from date of receipt when stored as instructed This protein is manufactured by Sino Biological Inc
    Catalog Number:
    10012h08h25
    Price:
    None
    Applications:
    Enzyme & Protein Activity Assays|Protein Assay Controls, Reference Standards & Accessories|Protein Assays and Analysis|Protein Biology
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher egfr
    Mechanistic characterization of miR-1254 and <t>CCAR1</t> 5′ UTR in modulating cellular sensitivity to tamoxifen. (A) miR-1254 targets predicted by TargetScan and miRanda with further GO analysis. NCOA1 , NCOA3 , <t>EGFR</t> , ERBB2 and SNAI1 are selected. (B) Relative luciferase activity of wild-type or mutant 3′ UTRs with forced expression of miR-1254. Error bars indicate SEM. *** P
    Kinase insert domain receptor KDR recombinant human protein is supplied as a lyophilized powder It is suitable for use in analysis of protein structure and performing cell based assays It can also be used as an immunogen as a protein standard or in other research applications This recombinant protein was expressed from a DNA sequence encoding the extracellular domain of human KDR NP 002244 1 Met 1 Glu 764 fused with a polyhistidine tag at the C terminus Activity Using the Octet RED System The affinity constant Kd of human KDR His bound to biotinylated human NRP2 VEGF165 was 5 9 nM Formulation Lyophilized in 140 mM NaCl 2 7 mM KCl 10 mM Na2HPO4 1 8 mM KH2PO4 pH 7 4 5 Mannitol 5 Trehalose 0 02 Tween 80 Reconstitution Dissolve the protein in sterile double distilled water to a concentration of 0 2 mg ml or lower It is recommended that the protein be aliquoted and be used as soon as possible Store aliquots under sterile conditions at 20°C Avoid repeated freeze thaw cycles Expiration Date Expires one year from date of receipt when stored as instructed This protein is manufactured by Sino Biological Inc
    https://www.bioz.com/result/egfr/product/Thermo Fisher
    Average 98 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    egfr - by Bioz Stars, 2020-11
    98/100 stars

    Images

    1) Product Images from "CCAR1 5′ UTR as a natural miRancer of miR-1254 overrides tamoxifen resistance"

    Article Title: CCAR1 5′ UTR as a natural miRancer of miR-1254 overrides tamoxifen resistance

    Journal: Cell Research

    doi: 10.1038/cr.2016.32

    Mechanistic characterization of miR-1254 and CCAR1 5′ UTR in modulating cellular sensitivity to tamoxifen. (A) miR-1254 targets predicted by TargetScan and miRanda with further GO analysis. NCOA1 , NCOA3 , EGFR , ERBB2 and SNAI1 are selected. (B) Relative luciferase activity of wild-type or mutant 3′ UTRs with forced expression of miR-1254. Error bars indicate SEM. *** P
    Figure Legend Snippet: Mechanistic characterization of miR-1254 and CCAR1 5′ UTR in modulating cellular sensitivity to tamoxifen. (A) miR-1254 targets predicted by TargetScan and miRanda with further GO analysis. NCOA1 , NCOA3 , EGFR , ERBB2 and SNAI1 are selected. (B) Relative luciferase activity of wild-type or mutant 3′ UTRs with forced expression of miR-1254. Error bars indicate SEM. *** P

    Techniques Used: Luciferase, Activity Assay, Mutagenesis, Expressing

    2) Product Images from "Molecular mechanism underlying the pharmacological interactions of the protein kinase C-β inhibitor enzastaurin and erlotinib in non-small cell lung cancer cells"

    Article Title: Molecular mechanism underlying the pharmacological interactions of the protein kinase C-β inhibitor enzastaurin and erlotinib in non-small cell lung cancer cells

    Journal: American Journal of Cancer Research

    doi:

    VEGFR-2 (A) and EGFR (B) mRNA expression as determined by real time RT-PCR after 72 hr treatment at IC 50 values of the drugs as specified in . Mean values ± SEM obtained from two independent experiments in triplicates. *: P
    Figure Legend Snippet: VEGFR-2 (A) and EGFR (B) mRNA expression as determined by real time RT-PCR after 72 hr treatment at IC 50 values of the drugs as specified in . Mean values ± SEM obtained from two independent experiments in triplicates. *: P

    Techniques Used: Expressing, Quantitative RT-PCR

    3) Product Images from "Evaluation of Gene Panel mRNAs in Urine Samples of Kidney Transplant Recipients as a Non-invasive Tool of Graft Function"

    Article Title: Evaluation of Gene Panel mRNAs in Urine Samples of Kidney Transplant Recipients as a Non-invasive Tool of Graft Function

    Journal:

    doi: 10.2119/2007-00017.Mas

    Receiver operating characteristic curves (ROC) for the better predictors. For each studied gene a ROC analysis predicting CAN versus SKF was performed. Afterward, the area under the ROC curve was estimated, A- TGF-β1 (AUC = 0.84); B- EGFR (AUC
    Figure Legend Snippet: Receiver operating characteristic curves (ROC) for the better predictors. For each studied gene a ROC analysis predicting CAN versus SKF was performed. Afterward, the area under the ROC curve was estimated, A- TGF-β1 (AUC = 0.84); B- EGFR (AUC

    Techniques Used:

    4) Product Images from "Effects of vascular endothelial growth factor and epidermal growth factor on biological properties of gastric cancer cells"

    Article Title: Effects of vascular endothelial growth factor and epidermal growth factor on biological properties of gastric cancer cells

    Journal: Archives of Medical Science : AMS

    doi: 10.5114/aoms.2019.88443

    mRNA expression of VEGF and EGF in cancer cells with or without Endostar or Erbitux treatment. A – mRNA levels of VEGF, EGF, EGFR, VEGFR1 and VEGFR2 in colon cancer cells: MGC803, HGC27, BGC823 and SGC7901. B – VEGF, VEGFR1 and VEGFR2 in MGC803 cancer cells treated with Endostar. C – VEGF, EGFR and EGF in MGC803 cancer cells treated with Erbitux. Quantification of band density is shown. VEGF and EGF mRNA levels were normalized to the β-actin mRNA level
    Figure Legend Snippet: mRNA expression of VEGF and EGF in cancer cells with or without Endostar or Erbitux treatment. A – mRNA levels of VEGF, EGF, EGFR, VEGFR1 and VEGFR2 in colon cancer cells: MGC803, HGC27, BGC823 and SGC7901. B – VEGF, VEGFR1 and VEGFR2 in MGC803 cancer cells treated with Endostar. C – VEGF, EGFR and EGF in MGC803 cancer cells treated with Erbitux. Quantification of band density is shown. VEGF and EGF mRNA levels were normalized to the β-actin mRNA level

    Techniques Used: Expressing

    5) Product Images from "Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding"

    Article Title: Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding

    Journal: The Biochemical journal

    doi: 10.1042/BCJ20160571

    R1166X mutation increases the amount of myosin VI in the cytosol pellet fraction. ( A ) RPE cells were transfected with WT full-length GFP-tagged myosin VI-NI, myosin VI R1166X and the rigor mutant, K157R and the distribution of myosin VI and loading controls, EGFR and actin, between supernatant (S) and pellet (P) was analysed by an actin pelleting assay followed by quantitative immunoblotting. ( B ) Quantitative immunoblotting of myosin VI distribution between pellet and supernatant fractions is shown (±SEM, n = 5, *** P
    Figure Legend Snippet: R1166X mutation increases the amount of myosin VI in the cytosol pellet fraction. ( A ) RPE cells were transfected with WT full-length GFP-tagged myosin VI-NI, myosin VI R1166X and the rigor mutant, K157R and the distribution of myosin VI and loading controls, EGFR and actin, between supernatant (S) and pellet (P) was analysed by an actin pelleting assay followed by quantitative immunoblotting. ( B ) Quantitative immunoblotting of myosin VI distribution between pellet and supernatant fractions is shown (±SEM, n = 5, *** P

    Techniques Used: Mutagenesis, Transfection

    6) Product Images from "IKKα regulates human keratinocyte migration through surveillance of the redox environment"

    Article Title: IKKα regulates human keratinocyte migration through surveillance of the redox environment

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.197343

    Oxidized IKKα increases EGF expression and EGFR activity. (A) ChIP assay in HEK001 cells with or without H 2 O 2 shows decreased IKKα binding to the EGF promoter following H 2 O 2 treatment, whereas RNA polymerase II (RNApol2) binding increases, indicative of increased EGF expression (two biological replicates, three technical replicates each). IP, immunoprecipitation; TSS, transcriptional start site; neg., negative control. (B) Transcriptional activation of EGF after H 2 O 2 treatment. (C) H 2 O 2 treatment (0.1 µM) increases EGF mRNA stability within 30 min and leads to subsequent destabilization. H 2 O 2 does not induce ovo-like zinc finger 2 ( OVOL2 ) transcription in HEK001 cells, which is known to be oxidation independent. (D) Increased ERK1/2 phosphorylation (pERK1/2) in HEK001 cells adjacent to the scratch margin and quantification of ERK1/2 phosphorylation. Trans., transmission. (E) H 2 O 2 -dependent phosphorylation of EGFR at residue Y1173 (pEGFR Y1173 ) after acute scratch injury of HEK001 cells. Intracellular recycling of EGFR phosphorylated at Y1173 in scratch margin keratinocytes (arrows, inset) shown by immunofluorescence studies. (F) Rapid phosphorylation of EGFR at residue Y1173 at the scratch margin in immunofluorescence studies in the absence of exogenous EGF. Immunostaining was also prevalent at the cell boundary (arrowhead). p.s., post scratch; 15ʹ, 15 min. (G) Wound keratinocyte migration model. High levels of H 2 O 2 may outcompete available antioxidant complexes and lead to non-specific oxidation and apoptosis, whereas low levels stimulate cysteine sulfenylation and migration. Sulfenylation of IKKα activates keratinocyte migration through IKKα-mediated regulation of EGF transcription and stability. Low H 2 O 2 levels also promote EGFR phosphorylation at Y1173. FOXO1 might regulate H 2 O 2 levels to promote migration. Scale bars: 100 µm. One-way ANOVA and Tukey's multiple comparison post hoc tests were utilized. Significance: * P
    Figure Legend Snippet: Oxidized IKKα increases EGF expression and EGFR activity. (A) ChIP assay in HEK001 cells with or without H 2 O 2 shows decreased IKKα binding to the EGF promoter following H 2 O 2 treatment, whereas RNA polymerase II (RNApol2) binding increases, indicative of increased EGF expression (two biological replicates, three technical replicates each). IP, immunoprecipitation; TSS, transcriptional start site; neg., negative control. (B) Transcriptional activation of EGF after H 2 O 2 treatment. (C) H 2 O 2 treatment (0.1 µM) increases EGF mRNA stability within 30 min and leads to subsequent destabilization. H 2 O 2 does not induce ovo-like zinc finger 2 ( OVOL2 ) transcription in HEK001 cells, which is known to be oxidation independent. (D) Increased ERK1/2 phosphorylation (pERK1/2) in HEK001 cells adjacent to the scratch margin and quantification of ERK1/2 phosphorylation. Trans., transmission. (E) H 2 O 2 -dependent phosphorylation of EGFR at residue Y1173 (pEGFR Y1173 ) after acute scratch injury of HEK001 cells. Intracellular recycling of EGFR phosphorylated at Y1173 in scratch margin keratinocytes (arrows, inset) shown by immunofluorescence studies. (F) Rapid phosphorylation of EGFR at residue Y1173 at the scratch margin in immunofluorescence studies in the absence of exogenous EGF. Immunostaining was also prevalent at the cell boundary (arrowhead). p.s., post scratch; 15ʹ, 15 min. (G) Wound keratinocyte migration model. High levels of H 2 O 2 may outcompete available antioxidant complexes and lead to non-specific oxidation and apoptosis, whereas low levels stimulate cysteine sulfenylation and migration. Sulfenylation of IKKα activates keratinocyte migration through IKKα-mediated regulation of EGF transcription and stability. Low H 2 O 2 levels also promote EGFR phosphorylation at Y1173. FOXO1 might regulate H 2 O 2 levels to promote migration. Scale bars: 100 µm. One-way ANOVA and Tukey's multiple comparison post hoc tests were utilized. Significance: * P

    Techniques Used: Expressing, Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Negative Control, Activation Assay, Transmission Assay, Immunofluorescence, Immunostaining, Migration

    7) Product Images from "An oxygen-regulated switch in the protein synthesis machinery"

    Article Title: An oxygen-regulated switch in the protein synthesis machinery

    Journal: Nature

    doi: 10.1038/nature11055

    RBM4 recruits HIF-2α to the 3′UTR for hypoxic translation a , Co-immunoprecipitation of HIF-2α in normoxia and hypoxia. WCL, whole cell lysate. b , RNA immunoprecipitation of HIF-2α and RBM4 in HIF-2α or RBM4 knockdown cells. IN, input; RN, RNase-treated. c , Effect of silencing HIF-2α or RBM4 on the hypoxic expression of a luciferase reporter fused to the 4295-4861 segment of the EGFR 3′UTR. d, Global translation rates in normoxic or hypoxic HIF-2α and/or RBM4 knockdown cells. e , Expression of a luciferase reporter containing a CGG to AAA mutation near RBM4 crosslinking sites (red arrows), or in an unrelated upstream region (uCGG). c and e , Columns, mean (n = 3); error bars, s.e.m. Significance of fold change (Student’s t test) is shown. Experiments performed in U87MG glioblastoma.
    Figure Legend Snippet: RBM4 recruits HIF-2α to the 3′UTR for hypoxic translation a , Co-immunoprecipitation of HIF-2α in normoxia and hypoxia. WCL, whole cell lysate. b , RNA immunoprecipitation of HIF-2α and RBM4 in HIF-2α or RBM4 knockdown cells. IN, input; RN, RNase-treated. c , Effect of silencing HIF-2α or RBM4 on the hypoxic expression of a luciferase reporter fused to the 4295-4861 segment of the EGFR 3′UTR. d, Global translation rates in normoxic or hypoxic HIF-2α and/or RBM4 knockdown cells. e , Expression of a luciferase reporter containing a CGG to AAA mutation near RBM4 crosslinking sites (red arrows), or in an unrelated upstream region (uCGG). c and e , Columns, mean (n = 3); error bars, s.e.m. Significance of fold change (Student’s t test) is shown. Experiments performed in U87MG glioblastoma.

    Techniques Used: Immunoprecipitation, Expressing, Luciferase, Mutagenesis

    HIF-2α activates EGFR mRNA translation by interacting with its 3′UTR a–b , Western blot and polysomal distribution of EGFR protein and mRNA in HIF-2α, HIF-1α or HIF-1β knockdown cells in the presence of actinomycin D. c , Polysomal distribution of HIF-2α and HIF-1α in hypoxia. d , RNA immunoprecipitation of HIF-1α and HIF-2α. IN, input; RN, RNase-treated. e , Dual luciferase assays in cells transfected with EGFR 3′UTR reporter constructs. Significance of fold change (Student’s t test) is shown. Columns, mean (n = 3); error bars, s.e.m. f , Global translation rates of transcription-incompetent cells expressing shRNA targeting HIF-2α or HIF-1α. Experiments performed in U87MG glioblastoma.
    Figure Legend Snippet: HIF-2α activates EGFR mRNA translation by interacting with its 3′UTR a–b , Western blot and polysomal distribution of EGFR protein and mRNA in HIF-2α, HIF-1α or HIF-1β knockdown cells in the presence of actinomycin D. c , Polysomal distribution of HIF-2α and HIF-1α in hypoxia. d , RNA immunoprecipitation of HIF-1α and HIF-2α. IN, input; RN, RNase-treated. e , Dual luciferase assays in cells transfected with EGFR 3′UTR reporter constructs. Significance of fold change (Student’s t test) is shown. Columns, mean (n = 3); error bars, s.e.m. f , Global translation rates of transcription-incompetent cells expressing shRNA targeting HIF-2α or HIF-1α. Experiments performed in U87MG glioblastoma.

    Techniques Used: Western Blot, Immunoprecipitation, Luciferase, Transfection, Construct, Expressing, shRNA

    HIF-2α/RBM4 recruits the m 7 -GTP cap via an interaction with eIF4E2 a , Dual luciferase assays in cells transfected with reporter constructs containing the 5′UTR of actin or lamin a/c with or without a 3′ rHRE. Significance of fold change (Student’s t test) is shown. Columns, mean (n = 3); error bars, s.e.m. b , Co-immunoprecipitation of HIF-2α and RBM4 in hypoxia. WCL, whole cell lysate. c , Capture assays using m 7 -GTP beads in hypoxic cell lysates depleted in eIF4E or eIF4E2. GTP, proteins dislodged from the beads by GTP. m 7 -GTP, proteins bound to m 7 -GTP beads after GTP wash. d , Western blot of total EGFR, PDGFRA, IGF1R, HIF-2α, eIF4E and eIF4E2 levels in eIF4E or eIF4E2 knockdown cells. e , Polysomal distribution of HIF-2α/RBM4 mRNA targets in hypoxic eIF4E2 knockdown cells. Experiments performed in U87MG glioblastoma.
    Figure Legend Snippet: HIF-2α/RBM4 recruits the m 7 -GTP cap via an interaction with eIF4E2 a , Dual luciferase assays in cells transfected with reporter constructs containing the 5′UTR of actin or lamin a/c with or without a 3′ rHRE. Significance of fold change (Student’s t test) is shown. Columns, mean (n = 3); error bars, s.e.m. b , Co-immunoprecipitation of HIF-2α and RBM4 in hypoxia. WCL, whole cell lysate. c , Capture assays using m 7 -GTP beads in hypoxic cell lysates depleted in eIF4E or eIF4E2. GTP, proteins dislodged from the beads by GTP. m 7 -GTP, proteins bound to m 7 -GTP beads after GTP wash. d , Western blot of total EGFR, PDGFRA, IGF1R, HIF-2α, eIF4E and eIF4E2 levels in eIF4E or eIF4E2 knockdown cells. e , Polysomal distribution of HIF-2α/RBM4 mRNA targets in hypoxic eIF4E2 knockdown cells. Experiments performed in U87MG glioblastoma.

    Techniques Used: Luciferase, Transfection, Construct, Immunoprecipitation, Western Blot

    An oxygen-regulated switch from eIF4E- to eIF4E2-dependent protein synthesis a , eIF4E and eIF4E2 polysome association in normoxia and hypoxia. b , Dual luciferase assays in normoxic (left) and hypoxic (right) cells transfected with constructs containing actin or EGFR 5′UTRs and EGFR rHREs. Assays were performed on cells treated with rapamycin, an inhibitor of mTOR and eIF4E, and in knockdown cells of eIF4E, eIF4E2, HIF-2α or RBM4 and in a co-eIF4E/HIF-2α knockdown. Significance of fold change (Student’s t test) is shown. P, parental; rapa, rapamycin. Columns, mean (n = 3); error bars, s.e.m. c , Global translation rates in normoxic or hypoxic eIF4E or eIF4E2 knockdown cells. Experiments performed in U87MG glioblastoma.
    Figure Legend Snippet: An oxygen-regulated switch from eIF4E- to eIF4E2-dependent protein synthesis a , eIF4E and eIF4E2 polysome association in normoxia and hypoxia. b , Dual luciferase assays in normoxic (left) and hypoxic (right) cells transfected with constructs containing actin or EGFR 5′UTRs and EGFR rHREs. Assays were performed on cells treated with rapamycin, an inhibitor of mTOR and eIF4E, and in knockdown cells of eIF4E, eIF4E2, HIF-2α or RBM4 and in a co-eIF4E/HIF-2α knockdown. Significance of fold change (Student’s t test) is shown. P, parental; rapa, rapamycin. Columns, mean (n = 3); error bars, s.e.m. c , Global translation rates in normoxic or hypoxic eIF4E or eIF4E2 knockdown cells. Experiments performed in U87MG glioblastoma.

    Techniques Used: Luciferase, Transfection, Construct

    8) Product Images from "Epidermal growth factor-receptor activation modulates Src-dependent resistance to lapatinib in breast cancer models"

    Article Title: Epidermal growth factor-receptor activation modulates Src-dependent resistance to lapatinib in breast cancer models

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr3650

    EGFR inhibition or silencing partially interferes with signal transduction in lapatinib-resistant breast cancer cells. (A) Western blot analysis of total cell lysates from MDA-MB-361, SKBR-3, MDA-MB-361-LR, and JIMT-1 human breast cancer cell lines grown in complete medium and treated with lapatinib (1 μ M ), cetuximab (0.35 μ M ), or the combination. (B) Western blot analysis on total cell lysates from MDA-MB-361, SKBR-3, MDA-MB-361-LR, and JIMT-1 human breast cancer cell lines transfected with an EGFR-specific siRNA (5 n M ) and treated or not with lapatinib (1 μ M ). The relative optical density of phospho-protein levels normalized to the actin level is shown.
    Figure Legend Snippet: EGFR inhibition or silencing partially interferes with signal transduction in lapatinib-resistant breast cancer cells. (A) Western blot analysis of total cell lysates from MDA-MB-361, SKBR-3, MDA-MB-361-LR, and JIMT-1 human breast cancer cell lines grown in complete medium and treated with lapatinib (1 μ M ), cetuximab (0.35 μ M ), or the combination. (B) Western blot analysis on total cell lysates from MDA-MB-361, SKBR-3, MDA-MB-361-LR, and JIMT-1 human breast cancer cell lines transfected with an EGFR-specific siRNA (5 n M ) and treated or not with lapatinib (1 μ M ). The relative optical density of phospho-protein levels normalized to the actin level is shown.

    Techniques Used: Inhibition, Transduction, Western Blot, Multiple Displacement Amplification, Transfection

    Src contributes to lapatinib resistance in breast cancer cells by interacting with EGFR rather than with HER2. (A) Western blot analysis of total cell lysates from MDA-MB-361, SKBR-3, MDA-MB-361-LR, and JIMT-1 human breast cancer cell lines transfected with an Src-specific siRNA (50 n M ) and treated or not with lapatinib (1 μ M ). The siRNA-negative control consists of nontargeting sequences. (B) Immunoprecipitation using anti-HER2, anti-Src, and anti-EGFR antibodies and blotting with anti-EGFR, -HER2, -HER3, and -Src antibodies of MDA-MB-361, SKBR-3, MDA-MB-361-LR, and JIMT-1 human breast cancer cells grown in complete medium. Total lysate, not immunoprecipitated, from BT474 cells was used as positive control. (C) Western blot analysis of total cell lysates from MDA-MB-361, SKBR-3, MDA-MB-361LR, and JIMT-1 human breast cancer cell lines grown in complete medium. The relative optical density of phospho-protein levels normalized to the actin level is shown.
    Figure Legend Snippet: Src contributes to lapatinib resistance in breast cancer cells by interacting with EGFR rather than with HER2. (A) Western blot analysis of total cell lysates from MDA-MB-361, SKBR-3, MDA-MB-361-LR, and JIMT-1 human breast cancer cell lines transfected with an Src-specific siRNA (50 n M ) and treated or not with lapatinib (1 μ M ). The siRNA-negative control consists of nontargeting sequences. (B) Immunoprecipitation using anti-HER2, anti-Src, and anti-EGFR antibodies and blotting with anti-EGFR, -HER2, -HER3, and -Src antibodies of MDA-MB-361, SKBR-3, MDA-MB-361-LR, and JIMT-1 human breast cancer cells grown in complete medium. Total lysate, not immunoprecipitated, from BT474 cells was used as positive control. (C) Western blot analysis of total cell lysates from MDA-MB-361, SKBR-3, MDA-MB-361LR, and JIMT-1 human breast cancer cell lines grown in complete medium. The relative optical density of phospho-protein levels normalized to the actin level is shown.

    Techniques Used: Western Blot, Multiple Displacement Amplification, Transfection, Negative Control, Immunoprecipitation, Positive Control

    9) Product Images from "Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model"

    Article Title: Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059597

    Fluorescence micrographs showing EGFR (Alexa 488; green), Rictor (Alexa 488; yellow) and cell nuclei (Hoechst 33342; blue) in GBM4 GBM-derived cancer stem-like cell line, and Gli36, U251MG, U118MG and LN229 GBM cell lines.
    Figure Legend Snippet: Fluorescence micrographs showing EGFR (Alexa 488; green), Rictor (Alexa 488; yellow) and cell nuclei (Hoechst 33342; blue) in GBM4 GBM-derived cancer stem-like cell line, and Gli36, U251MG, U118MG and LN229 GBM cell lines.

    Techniques Used: Fluorescence, Derivative Assay

    Transfection of siRNA sequences specific to Rictor and EGFR results in downregulation of their respective proteins in U251MG cell line. Optical density values shown are expressed relative to values obtained from untreated cells, which correspond to a value of 1. a ) Representative immunoblots showing ILK, Rictor, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against ILK or Rictor or the negative control sequence (Ng ctrl). b ) Representative immunoblots showing EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against EGFR or the negative control sequence (Neg ctrl). c ) Representative immunoblots showing Rictor, EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96 hrs after transfection of the combination of Rictor and EGFR siRNAs or the combination of two negative control sequences (Ng2x). Optical density values shown under each band represent the average obtained from three independent experiments (±SEM) normalized to β-actin, and AKT in the case of p(473)AKT. d ) Representative fluorescence photomicrograph (n = 3) of U251MG cells showing nuclei (Draq5; red), F-actin (Texas red phalloidin; Yellow), and p(473)-AKT (Alexa 488; blue) 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR, or the combination of two negative sequences (Ng2x).
    Figure Legend Snippet: Transfection of siRNA sequences specific to Rictor and EGFR results in downregulation of their respective proteins in U251MG cell line. Optical density values shown are expressed relative to values obtained from untreated cells, which correspond to a value of 1. a ) Representative immunoblots showing ILK, Rictor, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against ILK or Rictor or the negative control sequence (Ng ctrl). b ) Representative immunoblots showing EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against EGFR or the negative control sequence (Neg ctrl). c ) Representative immunoblots showing Rictor, EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96 hrs after transfection of the combination of Rictor and EGFR siRNAs or the combination of two negative control sequences (Ng2x). Optical density values shown under each band represent the average obtained from three independent experiments (±SEM) normalized to β-actin, and AKT in the case of p(473)AKT. d ) Representative fluorescence photomicrograph (n = 3) of U251MG cells showing nuclei (Draq5; red), F-actin (Texas red phalloidin; Yellow), and p(473)-AKT (Alexa 488; blue) 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR, or the combination of two negative sequences (Ng2x).

    Techniques Used: Transfection, Western Blot, Negative Control, Sequencing, Fluorescence

    Induction of lentiviral shRNA-transduced cells results in downregulation of corresponding proteins in vitro and downstream effectors, and reduction in cell migration. a ) Representative immunoblots showing Rictor, EGFR and β-actin from parental U251MG cells, U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor in the absence (-) or presence (+) of doxycycline. Average of band optical density normalized to β-actin from three independent experiments (+/−SEM), and expressed as relative to values obtained from parental cells, is shown under each band. *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to parental cells. b ) Representative immunoblots showing Rictor, EGFR, p(473)-AKT, p(346)-NDRG1, p(422)-SGK, p(657)-PKCα and β-actin from U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor exposed to doxycycline for 72hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251 Ng2x is shown under each band. c ) Representative immunoblots showing Rictor, p(473)-AKT and β-actin from U251 Rictor in the absence (-) of doxycycline or exposed to doxycycline for 24–120 hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251 Ng2x in the absence of doxycycline is shown under each band. d ) Scratch width scoring of U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor 18hrs after scratching in presence of doxycycline and after pre-incubation with doxycycline for 72 hrs.**p-value ≤0.01 compared to U251 Ng2x cells.
    Figure Legend Snippet: Induction of lentiviral shRNA-transduced cells results in downregulation of corresponding proteins in vitro and downstream effectors, and reduction in cell migration. a ) Representative immunoblots showing Rictor, EGFR and β-actin from parental U251MG cells, U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor in the absence (-) or presence (+) of doxycycline. Average of band optical density normalized to β-actin from three independent experiments (+/−SEM), and expressed as relative to values obtained from parental cells, is shown under each band. *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to parental cells. b ) Representative immunoblots showing Rictor, EGFR, p(473)-AKT, p(346)-NDRG1, p(422)-SGK, p(657)-PKCα and β-actin from U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor exposed to doxycycline for 72hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251 Ng2x is shown under each band. c ) Representative immunoblots showing Rictor, p(473)-AKT and β-actin from U251 Rictor in the absence (-) of doxycycline or exposed to doxycycline for 24–120 hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251 Ng2x in the absence of doxycycline is shown under each band. d ) Scratch width scoring of U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor 18hrs after scratching in presence of doxycycline and after pre-incubation with doxycycline for 72 hrs.**p-value ≤0.01 compared to U251 Ng2x cells.

    Techniques Used: shRNA, In Vitro, Migration, Western Blot, Incubation

    The combined silencing of Rictor and EGFR in vivo results in a complete inhibition of tumor growth. U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor cells were implanted into the right caudate nucleus-putamen of Rag2M mice (n = 6−8). Induction of shRNA expressi on in mice was initiated on day 21 by dissolving 2 mg/mL doxycyline and 5% sucrose in drinking water. a ) On day 49, animals were imaged by Maestro™ fluorescence imaging unit for the expression of tRFP co-expressed with the shRNA sequences upon doxycycline-induced expression. Mice were then terminated and brains were harvested, sectioned and stained for nuclei, Rictor, EGFR and p(473)-AKT and imaged for all markers in addition to tRFP by robotic fluorescence microscopy. No tumor was detected in the U251 EGFR/Rictor group. b ) A representative brain section from U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor tumor groups is shown: tRFP (red) and Hoechst (blue). c ) A representative tumor section from U251 Ng2x , U251 Rictor and U251 EGFR tumor groups is shown: nuclei (blue), rRFP (red), Rictor (yellow), EGFR (green) and p(473)-AKT (orange). d ) The expression of EGFR (left axis), Rictor (right axis) and p(473)-AKT (right axis) in U251 Ng2x , U251 Rictor , U251 EGFR tumor sections were quantified (positive staining normalized to Hoechst nuclei staining). e ) Tumor sizes were estimated by quantification of tumor areas in brain sections from all groups (left axis). The expression of the proliferation marker Ki67 in the tumor (proliferating fraction) was also quantified (right axis). *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to control untreated cells. ‡: No tumor was detected in the U251 EGFR/Rictor group.
    Figure Legend Snippet: The combined silencing of Rictor and EGFR in vivo results in a complete inhibition of tumor growth. U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor cells were implanted into the right caudate nucleus-putamen of Rag2M mice (n = 6−8). Induction of shRNA expressi on in mice was initiated on day 21 by dissolving 2 mg/mL doxycyline and 5% sucrose in drinking water. a ) On day 49, animals were imaged by Maestro™ fluorescence imaging unit for the expression of tRFP co-expressed with the shRNA sequences upon doxycycline-induced expression. Mice were then terminated and brains were harvested, sectioned and stained for nuclei, Rictor, EGFR and p(473)-AKT and imaged for all markers in addition to tRFP by robotic fluorescence microscopy. No tumor was detected in the U251 EGFR/Rictor group. b ) A representative brain section from U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor tumor groups is shown: tRFP (red) and Hoechst (blue). c ) A representative tumor section from U251 Ng2x , U251 Rictor and U251 EGFR tumor groups is shown: nuclei (blue), rRFP (red), Rictor (yellow), EGFR (green) and p(473)-AKT (orange). d ) The expression of EGFR (left axis), Rictor (right axis) and p(473)-AKT (right axis) in U251 Ng2x , U251 Rictor , U251 EGFR tumor sections were quantified (positive staining normalized to Hoechst nuclei staining). e ) Tumor sizes were estimated by quantification of tumor areas in brain sections from all groups (left axis). The expression of the proliferation marker Ki67 in the tumor (proliferating fraction) was also quantified (right axis). *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to control untreated cells. ‡: No tumor was detected in the U251 EGFR/Rictor group.

    Techniques Used: In Vivo, Inhibition, Mouse Assay, shRNA, Fluorescence, Imaging, Expressing, Staining, Microscopy, Marker

    Un-induced lentiviral shRNA-transduced cell lines behave similarly to each other. a ) Relative cell proliferation measured by MTT assay (24–96 hr time points) of U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor in the absence of doxycycline. Optical density values are normalized to values obtained at 24 hrs. b ) Fraction affected (normalized to untreated cells) measured by MTT assay of U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor in absence of doxycycline and treated with irinotecan, vincristine and temozolomide for 72 hrs. For in vitro data, all values shown represent the average from three independent experiments. c ) Subcutaneous tumor weight 14 days after inoculation of U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor in the absence of doxycycline (3 mice/group).
    Figure Legend Snippet: Un-induced lentiviral shRNA-transduced cell lines behave similarly to each other. a ) Relative cell proliferation measured by MTT assay (24–96 hr time points) of U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor in the absence of doxycycline. Optical density values are normalized to values obtained at 24 hrs. b ) Fraction affected (normalized to untreated cells) measured by MTT assay of U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor in absence of doxycycline and treated with irinotecan, vincristine and temozolomide for 72 hrs. For in vitro data, all values shown represent the average from three independent experiments. c ) Subcutaneous tumor weight 14 days after inoculation of U251 Ng2x , U251 Rictor , U251 EGFR and U251 EGFR/Rictor in the absence of doxycycline (3 mice/group).

    Techniques Used: shRNA, MTT Assay, In Vitro, Mouse Assay

    The combination of EGFR and Rictor silencing results in a reduction in cell migration. a ) Example of scoring chart for the scratch-wound healing assay. b ) Scratch width scoring of U251MG, U118MG and LN229 cells 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR or the combination of two negative control sequences (Ng2x), obtained from three independent experiments. ***p-value ≤0.001 compared to untreated cells.
    Figure Legend Snippet: The combination of EGFR and Rictor silencing results in a reduction in cell migration. a ) Example of scoring chart for the scratch-wound healing assay. b ) Scratch width scoring of U251MG, U118MG and LN229 cells 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR or the combination of two negative control sequences (Ng2x), obtained from three independent experiments. ***p-value ≤0.001 compared to untreated cells.

    Techniques Used: Migration, Wound Healing Assay, Transfection, Negative Control

    The combination of EGFR and Rictor silencing results in an increase in cell sensitivity to chemotherapeutic drugs. Changes in fraction affected measured by MTT assay of a ) U251MG, b ) U118MG and c ) LN229 cells 96 hrs after transfection of siRNA against Rictor, EGFR or the combination of Rictor and EGFR, and treated with irinotecan, vincristine or temozolomide at EC50 concentrations determined for control non-transfected cells at 72 hrs. Transfection of the two negative control sequences did induce an increase in Fa of LN229 exposed to vincristine of 0.13 compared to non-transfected cells and in this case only, changes in Fa are expressed relative to Ng2x-transfected cells. For all other cases, changes in Fa are expressed relative to control non-transfected cells. *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001.
    Figure Legend Snippet: The combination of EGFR and Rictor silencing results in an increase in cell sensitivity to chemotherapeutic drugs. Changes in fraction affected measured by MTT assay of a ) U251MG, b ) U118MG and c ) LN229 cells 96 hrs after transfection of siRNA against Rictor, EGFR or the combination of Rictor and EGFR, and treated with irinotecan, vincristine or temozolomide at EC50 concentrations determined for control non-transfected cells at 72 hrs. Transfection of the two negative control sequences did induce an increase in Fa of LN229 exposed to vincristine of 0.13 compared to non-transfected cells and in this case only, changes in Fa are expressed relative to Ng2x-transfected cells. For all other cases, changes in Fa are expressed relative to control non-transfected cells. *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001.

    Techniques Used: MTT Assay, Transfection, Negative Control

    Transfection of siRNA sequences specific to Rictor and EGFR results in downregulation of their respective proteins in U118MG and LN229 GBM lines. Histograms showing Rictor, p(473)AKT, AKT and β-actin protein levels relative to untreated cells. Optical density values were normalized to the β-actin value, and the AKT value in the case of p(473)AKT, and represent the average obtained from three independent experiments from a ) U118MG and b ) LN229 cells 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR siRNAs or the combination of two negative control sequences (Ng2x). *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001.
    Figure Legend Snippet: Transfection of siRNA sequences specific to Rictor and EGFR results in downregulation of their respective proteins in U118MG and LN229 GBM lines. Histograms showing Rictor, p(473)AKT, AKT and β-actin protein levels relative to untreated cells. Optical density values were normalized to the β-actin value, and the AKT value in the case of p(473)AKT, and represent the average obtained from three independent experiments from a ) U118MG and b ) LN229 cells 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR siRNAs or the combination of two negative control sequences (Ng2x). *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001.

    Techniques Used: Transfection, Negative Control

    10) Product Images from "EpCAM ectodomain EpEX is a ligand of EGFR that counteracts EGF-mediated epithelial-mesenchymal transition through modulation of phospho-ERK1/2 in head and neck cancers"

    Article Title: EpCAM ectodomain EpEX is a ligand of EGFR that counteracts EGF-mediated epithelial-mesenchymal transition through modulation of phospho-ERK1/2 in head and neck cancers

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2006624

    EGFR and EpCAM levels are molecular determinants of EMT induction, ERK activation, and migration. (A) Kyse30 cells were transfected with EGFR-specific siRNAs (pool of n = 4 siRNAs) (EGFR KD), siRNA controls (siRNA ctrl), EpCAM-specific shRNA (EpCAM KD) [ 54 ], control shRNA (shRNA ctrl), and a combination of EGFR siRNA and EpCAM shRNA (double KD). Expression of EGFR and EpCAM was assessed by immunoblotting with specific antibodies. Equal loading was confirmed by detecting actin levels. Clinical quadrants’ equivalents are indicated. Shown are representative results from n = 3 independent experiments. (B) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGF low (1.8 nM) and EGF high (9 nM). Cell morphology was assessed after 72 hr. Shown are representative images from n = 3 independent experiments. (C) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGF low (1.8 nM) for the indicated time points, and ERK1/2 phosphorylation was assessed by immunoblotting. Levels of ERK1/2 and actin were assessed in parallel. Shown are representative results from n = 3 independent experiments. (D) Quadrant 1 to 4 equivalents of Kyse30 cell variants were either kept untreated (control) or were treated with EGF low (1.8 nM). After 72 hr, mRNA transcript levels of Slug were assessed by qRT-PCR with specific primers. Shown are means with SDs from n = 2–3 independent experiments performed in triplicates. One-way ANOVA with post hoc multiple testing and Bonferroni correction * p-values
    Figure Legend Snippet: EGFR and EpCAM levels are molecular determinants of EMT induction, ERK activation, and migration. (A) Kyse30 cells were transfected with EGFR-specific siRNAs (pool of n = 4 siRNAs) (EGFR KD), siRNA controls (siRNA ctrl), EpCAM-specific shRNA (EpCAM KD) [ 54 ], control shRNA (shRNA ctrl), and a combination of EGFR siRNA and EpCAM shRNA (double KD). Expression of EGFR and EpCAM was assessed by immunoblotting with specific antibodies. Equal loading was confirmed by detecting actin levels. Clinical quadrants’ equivalents are indicated. Shown are representative results from n = 3 independent experiments. (B) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGF low (1.8 nM) and EGF high (9 nM). Cell morphology was assessed after 72 hr. Shown are representative images from n = 3 independent experiments. (C) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGF low (1.8 nM) for the indicated time points, and ERK1/2 phosphorylation was assessed by immunoblotting. Levels of ERK1/2 and actin were assessed in parallel. Shown are representative results from n = 3 independent experiments. (D) Quadrant 1 to 4 equivalents of Kyse30 cell variants were either kept untreated (control) or were treated with EGF low (1.8 nM). After 72 hr, mRNA transcript levels of Slug were assessed by qRT-PCR with specific primers. Shown are means with SDs from n = 2–3 independent experiments performed in triplicates. One-way ANOVA with post hoc multiple testing and Bonferroni correction * p-values

    Techniques Used: Activation Assay, Migration, Transfection, shRNA, Expressing, Quantitative RT-PCR

    EGFR and EpCAM expression predicts differential clinical outcome of HNSCCs. (A, C) Expression of EGFR and EpCAM was assessed in serial cryosections of normal mucosa and primary HNSCCs by IHC staining. Shown are representative examples of EGFR high /EpCAM high (A), EGFR high /EpCAM low , and EGFR low /EpCAM high (C) tumors at 100×, 200×, and 400× magnifications. (B) OS probability of HNSCC patients from the LMU cohort ( n = 180) and from a subcohort of the HNSCC TCGA cohort ( n = 279) [ 9 ] was stratified according to EGFR expression (cutoff threshold at the third quartile). IHC scores were generated for the LMU cohort as described [ 28 ]. Protein expression data for EGFR from the RPPA data were used for the TCGA cohort. OS is represented as Kaplan-Meier survival curves with p -value, HR, and CI for the entire cohorts and the HPV-negative subcohorts. Supporting data are compiled in S1 Data . (D, F) IHC scores of EGFR and EpCAM expression were assessed in n = 180 primary HNSCCs of the LMU cohort (D) and in n = 87 HPV-negative primary HNSCCs of the LMU cohort (F). Expression correlation of EGFR and EpCAM is plotted and subdivided according to a cutoff threshold of 150 (score range 0–300). Numbers and percentages of patients within subgroups are indicated in each quadrant. (E, G) OS and DFS were stratified according to all four quadrants defined in D and F and are represented as Kaplan-Meier survival curves with p -values, HRs, and CIs. Supporting data are compiled in S1 Data . CI, confidence interval; DFS, disease-free survival; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; HR, hazard ratio; IHC, immunohistochemistry; LMU, Ludwig-Maximilians-University; OS, overall survival; RPPA, reversed-phase protein atlas; TCGA, the Cancer Genome Atlas.
    Figure Legend Snippet: EGFR and EpCAM expression predicts differential clinical outcome of HNSCCs. (A, C) Expression of EGFR and EpCAM was assessed in serial cryosections of normal mucosa and primary HNSCCs by IHC staining. Shown are representative examples of EGFR high /EpCAM high (A), EGFR high /EpCAM low , and EGFR low /EpCAM high (C) tumors at 100×, 200×, and 400× magnifications. (B) OS probability of HNSCC patients from the LMU cohort ( n = 180) and from a subcohort of the HNSCC TCGA cohort ( n = 279) [ 9 ] was stratified according to EGFR expression (cutoff threshold at the third quartile). IHC scores were generated for the LMU cohort as described [ 28 ]. Protein expression data for EGFR from the RPPA data were used for the TCGA cohort. OS is represented as Kaplan-Meier survival curves with p -value, HR, and CI for the entire cohorts and the HPV-negative subcohorts. Supporting data are compiled in S1 Data . (D, F) IHC scores of EGFR and EpCAM expression were assessed in n = 180 primary HNSCCs of the LMU cohort (D) and in n = 87 HPV-negative primary HNSCCs of the LMU cohort (F). Expression correlation of EGFR and EpCAM is plotted and subdivided according to a cutoff threshold of 150 (score range 0–300). Numbers and percentages of patients within subgroups are indicated in each quadrant. (E, G) OS and DFS were stratified according to all four quadrants defined in D and F and are represented as Kaplan-Meier survival curves with p -values, HRs, and CIs. Supporting data are compiled in S1 Data . CI, confidence interval; DFS, disease-free survival; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; HR, hazard ratio; IHC, immunohistochemistry; LMU, Ludwig-Maximilians-University; OS, overall survival; RPPA, reversed-phase protein atlas; TCGA, the Cancer Genome Atlas.

    Techniques Used: Expressing, Immunohistochemistry, Staining, Generated

    Soluble EpEX-Fc binds to EGFR and induces ERK1/2 and AKT. (A) Bidirectional co-immunoprecipitation (“IP”) of EGFR and EpCAM in whole-cell lysates of FaDu, Cal27, and HCT8 cells using EGFR- and EpCAM-specific antibodies. Isotype control antibody (“IgG”) served as control. Coimmunoprecipitated EGFR and EpCAM were visualized in immunoblotting with specific antibodies (“IB”), with whole-cell lysates as control (“lysate”). Shown are representative results from n = 3 independent experiments. (B) SNs of Cal27, Kyse30, FaDu, and HCT8 cells were immunoprecipitated with EpEX-specific antibodies and separated under reducing (left) and nonreducing native conditions (right), and EpEX was detected with specific antibodies. Antibody HCs and EpEX mono-, di-, and oligomers are indicated. Shown are representative results from n = 3 independent experiments. (C) EpEX-Fc or Fc were incubated with whole-cell lysates of FaDu and Cal27 and immobilized on protein A agarose beads, and protein complexes were separated on SDS-PAGE. Immunoprecipitated proteins were detected by immunoblotting (“IB”) with Fc- and EGFR-specific antibodies. Shown are representative results from n = 3 independent experiments. (D) EGFR ex and EpEX were incubated in the presence or absence of cross-linker (BS3). Where indicated, EGF was added. Monomers, dimers, and EGFR ex /EpEX complexes are marked. Shown are representative results from n = 3 independent experiments. (E, F) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points. Where indicated, cells were additionally treated with Cetuximab (“Cet.”). Phosphorylation of ERK1/2 (E) and AKT (F) was assessed by immunoblotting with specific antibodies. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (G) FaDu and Cal27 cells were kept untreated or were treated with EGF (9 nM), Fc, or EpEX-Fc (10 nM) for 30 min, and phosphorylation of ERK1/2 and AKT was detected by immunofluorescence laser scanning confocal microscopy (ERK1/2 or AKT: green, nuclei: blue [DAPI]). Shown are representative results from n = 3 independent experiments. (H) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points (“EGF 30 min”). Where indicated, MEK1 inhibitor AZD6244 or EGFR inhibitor AG1478 were added. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (I) HEK293 cells were transiently transfected with GFP or EGFR expression plasmids and were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Expression of EGFR and activation of ERK1/2 were assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n = 3 independent experiments. (J) Expression of EpCAM and EGFR was assessed in HCT8WT and CRISPR-Cas9 EpCAM K.O.1 [ 48 ] by immunoblotting. Levels of actin were assessed in parallel. Shown are representative results from n = 3 independent experiments. (K) HCT8WT and CRISPR-Cas9 EpCAM K.O.1 cells were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Activation of ERK1/2 was assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n = 3 independent experiments. BS3, bisulfosuccinimidyl suberate; CRISPR-Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated 9; EGF, epidermal growth factor; EGFR, EGF receptor; EGFR ex , extracellular domain of EGFR; EpCAM, epithelial cell adhesion molecule; EpCAM K.O.1, EPCAM -knockout clone 1; EpEX, extracellular domain of EpCAM; EpICD, intracellular domain of EpCAM; ERK1/2, extracellular signal–regulated kinase 1/2; Fc, fragment crystallizable region; GFP, green fluorescent protein; HC, heavy chain; HCT8WT, HCT8 wild type; HEK293, human embryonic kidney 293; IgG, immunoglobulin G; MW, molecular mass; pAKT, phosphorylated AKT; pERK, phosphorylated ERK; SN, supernatant.
    Figure Legend Snippet: Soluble EpEX-Fc binds to EGFR and induces ERK1/2 and AKT. (A) Bidirectional co-immunoprecipitation (“IP”) of EGFR and EpCAM in whole-cell lysates of FaDu, Cal27, and HCT8 cells using EGFR- and EpCAM-specific antibodies. Isotype control antibody (“IgG”) served as control. Coimmunoprecipitated EGFR and EpCAM were visualized in immunoblotting with specific antibodies (“IB”), with whole-cell lysates as control (“lysate”). Shown are representative results from n = 3 independent experiments. (B) SNs of Cal27, Kyse30, FaDu, and HCT8 cells were immunoprecipitated with EpEX-specific antibodies and separated under reducing (left) and nonreducing native conditions (right), and EpEX was detected with specific antibodies. Antibody HCs and EpEX mono-, di-, and oligomers are indicated. Shown are representative results from n = 3 independent experiments. (C) EpEX-Fc or Fc were incubated with whole-cell lysates of FaDu and Cal27 and immobilized on protein A agarose beads, and protein complexes were separated on SDS-PAGE. Immunoprecipitated proteins were detected by immunoblotting (“IB”) with Fc- and EGFR-specific antibodies. Shown are representative results from n = 3 independent experiments. (D) EGFR ex and EpEX were incubated in the presence or absence of cross-linker (BS3). Where indicated, EGF was added. Monomers, dimers, and EGFR ex /EpEX complexes are marked. Shown are representative results from n = 3 independent experiments. (E, F) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points. Where indicated, cells were additionally treated with Cetuximab (“Cet.”). Phosphorylation of ERK1/2 (E) and AKT (F) was assessed by immunoblotting with specific antibodies. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (G) FaDu and Cal27 cells were kept untreated or were treated with EGF (9 nM), Fc, or EpEX-Fc (10 nM) for 30 min, and phosphorylation of ERK1/2 and AKT was detected by immunofluorescence laser scanning confocal microscopy (ERK1/2 or AKT: green, nuclei: blue [DAPI]). Shown are representative results from n = 3 independent experiments. (H) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points (“EGF 30 min”). Where indicated, MEK1 inhibitor AZD6244 or EGFR inhibitor AG1478 were added. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (I) HEK293 cells were transiently transfected with GFP or EGFR expression plasmids and were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Expression of EGFR and activation of ERK1/2 were assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n = 3 independent experiments. (J) Expression of EpCAM and EGFR was assessed in HCT8WT and CRISPR-Cas9 EpCAM K.O.1 [ 48 ] by immunoblotting. Levels of actin were assessed in parallel. Shown are representative results from n = 3 independent experiments. (K) HCT8WT and CRISPR-Cas9 EpCAM K.O.1 cells were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Activation of ERK1/2 was assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n = 3 independent experiments. BS3, bisulfosuccinimidyl suberate; CRISPR-Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated 9; EGF, epidermal growth factor; EGFR, EGF receptor; EGFR ex , extracellular domain of EGFR; EpCAM, epithelial cell adhesion molecule; EpCAM K.O.1, EPCAM -knockout clone 1; EpEX, extracellular domain of EpCAM; EpICD, intracellular domain of EpCAM; ERK1/2, extracellular signal–regulated kinase 1/2; Fc, fragment crystallizable region; GFP, green fluorescent protein; HC, heavy chain; HCT8WT, HCT8 wild type; HEK293, human embryonic kidney 293; IgG, immunoglobulin G; MW, molecular mass; pAKT, phosphorylated AKT; pERK, phosphorylated ERK; SN, supernatant.

    Techniques Used: Immunoprecipitation, Incubation, SDS Page, Immunofluorescence, Confocal Microscopy, Transfection, Expressing, Activation Assay, CRISPR, Knock-Out

    pERK and Slug are coexpressed and predict poor survival of HNSCC patients. (A) HNSCC tumors were stained for the expression of pERK1/2 and Slug in consecutive cryosections. Shown are 2 examples each of EGFR low /EpCAM high (patients 1 and 2), EGFR high /EpCAM low (patients 3 and 4), and HNSCCs at 100× and 200× magnification. (B) IHC scores of pERK and Slug were compared in EGFR low /EpCAM high ( n = 37) and EGFR high /EpCAM low ( n = 39) HNSCCs. Shown are IHC score values with mean (lines) and Student t test. ** p-value
    Figure Legend Snippet: pERK and Slug are coexpressed and predict poor survival of HNSCC patients. (A) HNSCC tumors were stained for the expression of pERK1/2 and Slug in consecutive cryosections. Shown are 2 examples each of EGFR low /EpCAM high (patients 1 and 2), EGFR high /EpCAM low (patients 3 and 4), and HNSCCs at 100× and 200× magnification. (B) IHC scores of pERK and Slug were compared in EGFR low /EpCAM high ( n = 37) and EGFR high /EpCAM low ( n = 39) HNSCCs. Shown are IHC score values with mean (lines) and Student t test. ** p-value

    Techniques Used: Staining, Expressing, Immunohistochemistry

    Schematic representation of EGF and EpEX cross talk at the EGFR. Low-dose EGF induces EGFR activation that results in intermediate ERK1/2 phosphorylation and enhanced cell proliferation (left panel). High-dose EGF induces EGFR activation that results in strong ERK1/2 phosphorylation and induction of EMT, including EMT-TFs Snail, Zeb1, and Slug (center-left panel). High-dose EpEX induces EGFR activation that results in intermediate ERK1/2 phosphorylation and enhanced proliferation (center-right panel); low-dose EpEX has no measurable effect on proliferation (right panel). EGF, epidermal growth factor; EGFR, EGF receptor; EMT, epithelial-mesenchymal transition; EMT-TF, EMT transcription factor; EpEX, extracellular domain of EpCAM; ERK1/2, extracellular signal–regulated kinase; PI3K, phosphoinositide 3-kinase; Zeb1, zinc finger E-box-binding homeobox 1.
    Figure Legend Snippet: Schematic representation of EGF and EpEX cross talk at the EGFR. Low-dose EGF induces EGFR activation that results in intermediate ERK1/2 phosphorylation and enhanced cell proliferation (left panel). High-dose EGF induces EGFR activation that results in strong ERK1/2 phosphorylation and induction of EMT, including EMT-TFs Snail, Zeb1, and Slug (center-left panel). High-dose EpEX induces EGFR activation that results in intermediate ERK1/2 phosphorylation and enhanced proliferation (center-right panel); low-dose EpEX has no measurable effect on proliferation (right panel). EGF, epidermal growth factor; EGFR, EGF receptor; EMT, epithelial-mesenchymal transition; EMT-TF, EMT transcription factor; EpEX, extracellular domain of EpCAM; ERK1/2, extracellular signal–regulated kinase; PI3K, phosphoinositide 3-kinase; Zeb1, zinc finger E-box-binding homeobox 1.

    Techniques Used: Activation Assay, Binding Assay

    EpEX-Fc induces EGFR-dependent proliferation but inhibits high-dose EGF-induced EMT. (A) FaDu and Kyse30 cells were plated at equal numbers and treated with control media, low (1.8 nM) and high (9 nM) doses of EGF, low (1 nM) and high doses (10 nM) of EpEX-Fc, Fc (10 nM), or a combination of EpEX high with Cetuximab (“Cet.”). Cell numbers were assessed after 24, 48, and 72 hr. Shown are means with SDs from n = 3 independent experiments. Two-way ANOVA with post hoc multiple testing and Bonferroni correction. * 0.05, ** 0.01; *** 0.001; **** 0.0001. Supporting data are compiled in S1 Data . (B) FaDu and Kyse30 cells were plated at equal numbers and treated with control media, EpEX (10 nM), or a combination of EpEX (10 nM) and Cetuximab (“Cet.”). BrdU incorporation was analyzed after 72 hr. Shown are means with SDs from n = 3 independent experiments. Two-way ANOVA with post hoc multiple testing and Bonferroni correction. * 0.05. Supporting data are compiled in S1 Data . (C) Relative migration of FaDu and Kyse30 cells was assessed in wound healing assays. FaDu, Kyse30, and Cal27 cells were either kept untreated (control) or were treated with Fc (10 nM), EpEX-Fc, EGF, EGF in combination with EpEX (10 nM), or EGF with Cetuximab (“Cet.”). Shown are representative micrograph pictures of cells after 24 hr (Kyse30) and 48 hr (FaDu) from n = 3 independent experiments. (D) Relative migration was quantified from representative micrographs and was normalized for proliferation indexes of each cell line. Shown are means with SDs from n = 3 independent experiments. One-way ANOVA with post hoc multiple testing and Bonferroni correction * p -value
    Figure Legend Snippet: EpEX-Fc induces EGFR-dependent proliferation but inhibits high-dose EGF-induced EMT. (A) FaDu and Kyse30 cells were plated at equal numbers and treated with control media, low (1.8 nM) and high (9 nM) doses of EGF, low (1 nM) and high doses (10 nM) of EpEX-Fc, Fc (10 nM), or a combination of EpEX high with Cetuximab (“Cet.”). Cell numbers were assessed after 24, 48, and 72 hr. Shown are means with SDs from n = 3 independent experiments. Two-way ANOVA with post hoc multiple testing and Bonferroni correction. * 0.05, ** 0.01; *** 0.001; **** 0.0001. Supporting data are compiled in S1 Data . (B) FaDu and Kyse30 cells were plated at equal numbers and treated with control media, EpEX (10 nM), or a combination of EpEX (10 nM) and Cetuximab (“Cet.”). BrdU incorporation was analyzed after 72 hr. Shown are means with SDs from n = 3 independent experiments. Two-way ANOVA with post hoc multiple testing and Bonferroni correction. * 0.05. Supporting data are compiled in S1 Data . (C) Relative migration of FaDu and Kyse30 cells was assessed in wound healing assays. FaDu, Kyse30, and Cal27 cells were either kept untreated (control) or were treated with Fc (10 nM), EpEX-Fc, EGF, EGF in combination with EpEX (10 nM), or EGF with Cetuximab (“Cet.”). Shown are representative micrograph pictures of cells after 24 hr (Kyse30) and 48 hr (FaDu) from n = 3 independent experiments. (D) Relative migration was quantified from representative micrographs and was normalized for proliferation indexes of each cell line. Shown are means with SDs from n = 3 independent experiments. One-way ANOVA with post hoc multiple testing and Bonferroni correction * p -value

    Techniques Used: BrdU Incorporation Assay, Migration

    11) Product Images from "Identification of new candidate therapeutic target genes in head and neck squamous cell carcinomas"

    Article Title: Identification of new candidate therapeutic target genes in head and neck squamous cell carcinomas

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10163

    Normal and overexpressed tumors at the protein and mRNA levels for EGFR , MET and CDK6 . Immunohistochemical staining for EGFR ( A , B ), MET ( C , D ) and CDK6 ( E , F ) proteins in HNSCC tumors. Examples of three tumors with EGFR (A), MET (C) and CDK6 (E) normal mRNA-expressions and three tumors with EGFR (B), MET (D) and CDK6 (F) mRNA-overexpressions. Intense EGFR (B), MET (D) and CDK6 (F) immunoreactivity was found in tumor epithelial cells from the EGFR, MET, CK6 mRNA-overexpressing tumors but not in cells from the tumor without EGFR, MET, CK6 mRNA-overexpression (A, C, E) (original magnification × 50).
    Figure Legend Snippet: Normal and overexpressed tumors at the protein and mRNA levels for EGFR , MET and CDK6 . Immunohistochemical staining for EGFR ( A , B ), MET ( C , D ) and CDK6 ( E , F ) proteins in HNSCC tumors. Examples of three tumors with EGFR (A), MET (C) and CDK6 (E) normal mRNA-expressions and three tumors with EGFR (B), MET (D) and CDK6 (F) mRNA-overexpressions. Intense EGFR (B), MET (D) and CDK6 (F) immunoreactivity was found in tumor epithelial cells from the EGFR, MET, CK6 mRNA-overexpressing tumors but not in cells from the tumor without EGFR, MET, CK6 mRNA-overexpression (A, C, E) (original magnification × 50).

    Techniques Used: Immunohistochemistry, Staining, Over Expression

    12) Product Images from "Evaluation of the prognostic significance of HER family mRNA expression in high-risk early breast cancer: a Hellenic Cooperative Oncology Group (HeCOG) validation study"

    Article Title: Evaluation of the prognostic significance of HER family mRNA expression in high-risk early breast cancer: a Hellenic Cooperative Oncology Group (HeCOG) validation study

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-015-0530-0

    Scattered plots of mRNA expression values (40-DCT) of different HER family members. a HER2 vs. HER3; b HER2 vs. HER4; c HER3 vs. EGFR; and d HER3 vs. HER4. CT cycle threshold, DCT delta CT.
    Figure Legend Snippet: Scattered plots of mRNA expression values (40-DCT) of different HER family members. a HER2 vs. HER3; b HER2 vs. HER4; c HER3 vs. EGFR; and d HER3 vs. HER4. CT cycle threshold, DCT delta CT.

    Techniques Used: Expressing

    13) Product Images from "Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma"

    Article Title: Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06258-2

    Validation of TEAD1-binding targets by chromatin immunoprecipitation. a Density plot of correspondence analysis between chromatin accessibility and gene expression. Plotted on the y -axis is the average rld-normalized gene expression for each gene from all RNA-seq E+GSC sample data. Plotted on the x -axis is the highest ATAC-seq peak associated with the proximal promoter [−5 kb, +3 kb] of the same gene. Color intensity indicates density of the gene population, with red representing higher densities and blue representing lower densities. Strong correspondence is observed between open chromatin peaks and a moderate/high level of gene expression in all E+GSC samples (plot for one representative sample shown here). Several putative TEAD1-target genes of interest are indicated on the plot. b IGV plot of ATAC-seq piled reads at EGFR , AQP4 , and CDH4 in four different acutely sorted E+GSCs (D.PROM distal promoter of EGFR, peak180759: chr7:55,000,372–55,001,595; P.PROM proximal promoter to TSS of EGFR, peak180777: chr7:55,085,981–55,088,747). For this IGV representation, reads are centered on the cut site of the Tn5 enzyme, correcting for the 9 bp occupancy of Tn5, and presumed footprints/peak troughs corresponding to TEAD motifs are delineated by downward arrow. c Chromatin immunoprecipitation (ChIP-PCR) in GBM tissues. Significant enrichment of TEAD1 (but not TEAD4) over IgG is seen specifically at differential open chromatin peaks with associated TEAD1 motif within the upstream promoter of EGFR (D. PROM, chr7:55001141 motif start site) ( n = 6, * p = 0.011), at CDH4 (chr20:60011935 motif start site) ( n = 6, * p = 0.027), and at the proximal promoter of AQP4 (chr18:24444251 motif start site) ( n = 6, * p = 0.029). Significant binding above background is not observed at EGFR P.PROM or at chromatin inaccessible regions (IN2 = EGFR intron 2). Enrichment is expressed as fold increase over IgG, after normalization with 10% input, using 2^–ΔΔCt analysis accounting for primer efficiency: ( E ^IA− S ) sample /( E ^IA− S ) IgG . E : primer efficiency; IA: 10% Input-Adjusted Ct; S : sample Ct. Bars represent mean ± SEM. d Western immunoblot illustrates decreased expression of AQP4 and CDH4 in TEAD1KO but not in TEAD4KO G-13063 GBM cells
    Figure Legend Snippet: Validation of TEAD1-binding targets by chromatin immunoprecipitation. a Density plot of correspondence analysis between chromatin accessibility and gene expression. Plotted on the y -axis is the average rld-normalized gene expression for each gene from all RNA-seq E+GSC sample data. Plotted on the x -axis is the highest ATAC-seq peak associated with the proximal promoter [−5 kb, +3 kb] of the same gene. Color intensity indicates density of the gene population, with red representing higher densities and blue representing lower densities. Strong correspondence is observed between open chromatin peaks and a moderate/high level of gene expression in all E+GSC samples (plot for one representative sample shown here). Several putative TEAD1-target genes of interest are indicated on the plot. b IGV plot of ATAC-seq piled reads at EGFR , AQP4 , and CDH4 in four different acutely sorted E+GSCs (D.PROM distal promoter of EGFR, peak180759: chr7:55,000,372–55,001,595; P.PROM proximal promoter to TSS of EGFR, peak180777: chr7:55,085,981–55,088,747). For this IGV representation, reads are centered on the cut site of the Tn5 enzyme, correcting for the 9 bp occupancy of Tn5, and presumed footprints/peak troughs corresponding to TEAD motifs are delineated by downward arrow. c Chromatin immunoprecipitation (ChIP-PCR) in GBM tissues. Significant enrichment of TEAD1 (but not TEAD4) over IgG is seen specifically at differential open chromatin peaks with associated TEAD1 motif within the upstream promoter of EGFR (D. PROM, chr7:55001141 motif start site) ( n = 6, * p = 0.011), at CDH4 (chr20:60011935 motif start site) ( n = 6, * p = 0.027), and at the proximal promoter of AQP4 (chr18:24444251 motif start site) ( n = 6, * p = 0.029). Significant binding above background is not observed at EGFR P.PROM or at chromatin inaccessible regions (IN2 = EGFR intron 2). Enrichment is expressed as fold increase over IgG, after normalization with 10% input, using 2^–ΔΔCt analysis accounting for primer efficiency: ( E ^IA− S ) sample /( E ^IA− S ) IgG . E : primer efficiency; IA: 10% Input-Adjusted Ct; S : sample Ct. Bars represent mean ± SEM. d Western immunoblot illustrates decreased expression of AQP4 and CDH4 in TEAD1KO but not in TEAD4KO G-13063 GBM cells

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Expressing, RNA Sequencing Assay, Polymerase Chain Reaction, IA, Western Blot

    TEAD1 regulates expression of EGFR. a Immunofluorescence image depicts observed expression of EGFR in sham and TEAD1-knockout G-13063 xenografts 3.5 months post transplantation. Scale bar = 50 μM. b Western immunoblot depicts marked downregulation of EGFR in vitro after knockout of TEAD1, but not TEAD4, in G-13063 cells, which is partially restored after TEAD1 overexpression for 48 h. On right is a bar graph quantification of immunoblots from three independent experiments ( n = 3; TEAD1KO vs. TEAD4KO: ** p = 0.004 and ** p = 0.003 for TEAD1 and EGFR, respectively; TEAD1KO + TEAD1OE vs. TEAD1KO: * p = 0.01 for TEAD1; p = 0.068 for EGFR in one tail t -test analysis. Bars represent mean ± SEM). c Western immunoblot depicts downregulation of pERK/ERK but not pAKT/AKT in TEAD1KO cells, compared to sham ( n = 3 cell lines; pERK/ERK: * p = 0.029; bars represent mean ± SEM). On right are shown representative immunoblot images from one cell line
    Figure Legend Snippet: TEAD1 regulates expression of EGFR. a Immunofluorescence image depicts observed expression of EGFR in sham and TEAD1-knockout G-13063 xenografts 3.5 months post transplantation. Scale bar = 50 μM. b Western immunoblot depicts marked downregulation of EGFR in vitro after knockout of TEAD1, but not TEAD4, in G-13063 cells, which is partially restored after TEAD1 overexpression for 48 h. On right is a bar graph quantification of immunoblots from three independent experiments ( n = 3; TEAD1KO vs. TEAD4KO: ** p = 0.004 and ** p = 0.003 for TEAD1 and EGFR, respectively; TEAD1KO + TEAD1OE vs. TEAD1KO: * p = 0.01 for TEAD1; p = 0.068 for EGFR in one tail t -test analysis. Bars represent mean ± SEM). c Western immunoblot depicts downregulation of pERK/ERK but not pAKT/AKT in TEAD1KO cells, compared to sham ( n = 3 cell lines; pERK/ERK: * p = 0.029; bars represent mean ± SEM). On right are shown representative immunoblot images from one cell line

    Techniques Used: Expressing, Immunofluorescence, Knock-Out, Transplantation Assay, Western Blot, In Vitro, Over Expression

    14) Product Images from "Characteristics of Head and Neck Squamous Cell Carcinoma Cell Lines Reflect Human Tumor Biology Independent of Primary Etiologies and HPV Status"

    Article Title: Characteristics of Head and Neck Squamous Cell Carcinoma Cell Lines Reflect Human Tumor Biology Independent of Primary Etiologies and HPV Status

    Journal: Translational Oncology

    doi: 10.1016/j.tranon.2020.100808

    Biomarker expression by Western blotting during exponential growth phase. TP53 (A), RB1 (B), p16 (C), E6 (D), E7 (E), EGFR (F), Cyclin D1 (G), Ki-67 (H), total beta-catenin (I), and active beta-catenin (J). Protein was loaded at 50 μg per lane for all biomarkers except for total beta-catenin and active beta-catenin, which were loaded with 25 μg of protein. GAPDH was included as a loading control. (C and F) The same GAPDH staining was used since p16 and EGFR were probed from the same blot. (D) E6 was the only protein that was immunoprecipitated prior to WB due to its weaker expression. As a result, it was not possible to show a loading control. E6 was probed both in early (days 5-6) and later exponential growth phase (days 9-10, indicated by *). (G) Cyclin D1 staining was stripped to detect GAPDH.
    Figure Legend Snippet: Biomarker expression by Western blotting during exponential growth phase. TP53 (A), RB1 (B), p16 (C), E6 (D), E7 (E), EGFR (F), Cyclin D1 (G), Ki-67 (H), total beta-catenin (I), and active beta-catenin (J). Protein was loaded at 50 μg per lane for all biomarkers except for total beta-catenin and active beta-catenin, which were loaded with 25 μg of protein. GAPDH was included as a loading control. (C and F) The same GAPDH staining was used since p16 and EGFR were probed from the same blot. (D) E6 was the only protein that was immunoprecipitated prior to WB due to its weaker expression. As a result, it was not possible to show a loading control. E6 was probed both in early (days 5-6) and later exponential growth phase (days 9-10, indicated by *). (G) Cyclin D1 staining was stripped to detect GAPDH.

    Techniques Used: Biomarker Assay, Expressing, Western Blot, Staining, Immunoprecipitation

    15) Product Images from "CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression"

    Article Title: CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006030

    Expression of CCHCR1 in Bowen's disease, psoriasis, and actinic keratosis. Sections of Bowen's disease (A–D) immunostained with antibodies against CCHCR1 and EGFR as indicated. Serial sections of psoriasis (E, F) were stained with antibodies for CCHCR1 and Ki67. AK samples (G, H) were stained with CCHCR1 antibodies. In Bowen's disease (A, B), spongiosis, inflammatory infiltrate, and capillary proliferation are associated with CCHCR1 expression while areas lacking inflammation and spongiosis express less CCHCR1 (C). EGFR expression (D) in basal and suprabasal KCs of dermal papillae associates with CCHCR1 expression (B). Also in psoriasis (E), CCHCR1 is expressed basally or suprabasally (arrows) overlying the dermal papillae (dp) while rete ridges (rr) projecting into the dermis are almost negative for CCHCR1. In contrast, Ki67 expression (F) localizes to regions that are almost negative for CCHCR1 cells (arrowheads). AK samples (G) express CCHCR1 basally and suprabasally in association with KC heterogeneity, spongiosis or inflammation but adjacent areas with less inflammation or spongiosis (H) stain only faintly. Scale bars: (A–D) 50 µm; (E, F) 25 µm; (G, H) 12.5 µm.
    Figure Legend Snippet: Expression of CCHCR1 in Bowen's disease, psoriasis, and actinic keratosis. Sections of Bowen's disease (A–D) immunostained with antibodies against CCHCR1 and EGFR as indicated. Serial sections of psoriasis (E, F) were stained with antibodies for CCHCR1 and Ki67. AK samples (G, H) were stained with CCHCR1 antibodies. In Bowen's disease (A, B), spongiosis, inflammatory infiltrate, and capillary proliferation are associated with CCHCR1 expression while areas lacking inflammation and spongiosis express less CCHCR1 (C). EGFR expression (D) in basal and suprabasal KCs of dermal papillae associates with CCHCR1 expression (B). Also in psoriasis (E), CCHCR1 is expressed basally or suprabasally (arrows) overlying the dermal papillae (dp) while rete ridges (rr) projecting into the dermis are almost negative for CCHCR1. In contrast, Ki67 expression (F) localizes to regions that are almost negative for CCHCR1 cells (arrowheads). AK samples (G) express CCHCR1 basally and suprabasally in association with KC heterogeneity, spongiosis or inflammation but adjacent areas with less inflammation or spongiosis (H) stain only faintly. Scale bars: (A–D) 50 µm; (E, F) 25 µm; (G, H) 12.5 µm.

    Techniques Used: Expressing, Staining

    Expression of CCHCR1, EGFR, and Ki67 in keratoacanthoma. Serial sections of KA (A, B, E, F) were stained with the antibodies against CCHCR1 and EGFR (A, B) or against CCHCR1 and Ki67 (E, F). Higher magnifications of A and B are shown (C, D, respectively). The pushing border of a KA is CCHCR1 positive (A) co-localizing with EGFR (B). Another sample is shown (E) with a prominent lymphocyte infiltrate and cytoplasmic CCHCR1 expression of the pushing border cells. More scarce Ki67 expression (F) is defined to the same areas but not necessarily to the same cells. Arrows indicate corresponding positions. Scale bars: (A, B, E, F) 50 µm; (C, D) 12.5 µm.
    Figure Legend Snippet: Expression of CCHCR1, EGFR, and Ki67 in keratoacanthoma. Serial sections of KA (A, B, E, F) were stained with the antibodies against CCHCR1 and EGFR (A, B) or against CCHCR1 and Ki67 (E, F). Higher magnifications of A and B are shown (C, D, respectively). The pushing border of a KA is CCHCR1 positive (A) co-localizing with EGFR (B). Another sample is shown (E) with a prominent lymphocyte infiltrate and cytoplasmic CCHCR1 expression of the pushing border cells. More scarce Ki67 expression (F) is defined to the same areas but not necessarily to the same cells. Arrows indicate corresponding positions. Scale bars: (A, B, E, F) 50 µm; (C, D) 12.5 µm.

    Techniques Used: Expressing, Staining

    Expression and regulation of CCHCR1, Ki67, and EGFR mRNA in HaCaT cells, its ras-transformed clones and SCC cell lines. CCHCR1 (A), Ki67 (B), and EGFR (C) mRNA expression levels (TaqMan) in HaCaT cells after treatment with the tumor promoters OA and menadione. CCHCR1 (D), Ki67 (E) and EGFR (F) mRNA expression in HaCaT, A5, II4, RT3, A431, and FaDu cells. The invasive cells II4 and FaDu and metastatic RT3 cells expressed less CCHCR1 (D) and Ki67 (E) than HaCaT and A5 cells. A431 cells (F) express EGFR mRNA clearly more than other cell lines. The ras-transformed clones A5, II4, and RT3 and FaDu cells express EGFR less than HaCaT cells. Quantitative RT-PCR results are shown relative to mRNA levels from corresponding control cells (assigned the value 1). Expression levels of CCHCR1, Ki67, and EGFR in HaCaT cells were normalized to the GAPDH mRNA levels in the same samples. * p
    Figure Legend Snippet: Expression and regulation of CCHCR1, Ki67, and EGFR mRNA in HaCaT cells, its ras-transformed clones and SCC cell lines. CCHCR1 (A), Ki67 (B), and EGFR (C) mRNA expression levels (TaqMan) in HaCaT cells after treatment with the tumor promoters OA and menadione. CCHCR1 (D), Ki67 (E) and EGFR (F) mRNA expression in HaCaT, A5, II4, RT3, A431, and FaDu cells. The invasive cells II4 and FaDu and metastatic RT3 cells expressed less CCHCR1 (D) and Ki67 (E) than HaCaT and A5 cells. A431 cells (F) express EGFR mRNA clearly more than other cell lines. The ras-transformed clones A5, II4, and RT3 and FaDu cells express EGFR less than HaCaT cells. Quantitative RT-PCR results are shown relative to mRNA levels from corresponding control cells (assigned the value 1). Expression levels of CCHCR1, Ki67, and EGFR in HaCaT cells were normalized to the GAPDH mRNA levels in the same samples. * p

    Techniques Used: Expressing, Transformation Assay, Clone Assay, Quantitative RT-PCR

    Expression of CCHCR1, Ki67, and EGFR in HaCaT cell proliferation assay. A) Relative expression of CCHCR1 mRNA (as measured by TaqMan) at different time points (from 24 h to 8 d) after releasing the cells from serum starvation and high density culturing. Expression of CCHCR1 did not differ between confluent (control) cells and quiescent (starved) cells. Control cells expressed CCHCR1 2.5-fold more than proliferative HaCaT cells (24–48 h, 3–4 d). As confluency was reattained (8 d), the expression of CCHCR1 increased to the level of the control cells. B) Correlation between relative CCHCR1 and Ki67 expression levels. When CCHCR1 mRNA expression levels were compared to those of Ki67, a negative correlation was seen, confirming the proliferative status of the HaCaT cells. C) EGFR mRNA expression correlated with CCHCR1 mRNA expression. D) Correlation between CCHCR1 and Ki67 expression in the experiment with lower cell density. The negative correlation of CCHCR1 expression with Ki67 expression was even more profound as relative CCHCR1 mRNA decreased near to zero in the two control cells. E) Correlation between CCHCR1 and EGFR expression in the experiment with lower cell density. Here again, CCHCR1 expression correlated with EGFR expression. TaqMan PCR results are shown relative to mRNA levels from corresponding control cells assigned the value 1. Expression levels of CCHCR1, Ki67, and EGFR in HaCaT cells were normalized to the GAPDH mRNA levels in the same samples. * p
    Figure Legend Snippet: Expression of CCHCR1, Ki67, and EGFR in HaCaT cell proliferation assay. A) Relative expression of CCHCR1 mRNA (as measured by TaqMan) at different time points (from 24 h to 8 d) after releasing the cells from serum starvation and high density culturing. Expression of CCHCR1 did not differ between confluent (control) cells and quiescent (starved) cells. Control cells expressed CCHCR1 2.5-fold more than proliferative HaCaT cells (24–48 h, 3–4 d). As confluency was reattained (8 d), the expression of CCHCR1 increased to the level of the control cells. B) Correlation between relative CCHCR1 and Ki67 expression levels. When CCHCR1 mRNA expression levels were compared to those of Ki67, a negative correlation was seen, confirming the proliferative status of the HaCaT cells. C) EGFR mRNA expression correlated with CCHCR1 mRNA expression. D) Correlation between CCHCR1 and Ki67 expression in the experiment with lower cell density. The negative correlation of CCHCR1 expression with Ki67 expression was even more profound as relative CCHCR1 mRNA decreased near to zero in the two control cells. E) Correlation between CCHCR1 and EGFR expression in the experiment with lower cell density. Here again, CCHCR1 expression correlated with EGFR expression. TaqMan PCR results are shown relative to mRNA levels from corresponding control cells assigned the value 1. Expression levels of CCHCR1, Ki67, and EGFR in HaCaT cells were normalized to the GAPDH mRNA levels in the same samples. * p

    Techniques Used: Expressing, Proliferation Assay, Polymerase Chain Reaction

    CCHCR1 mRNA expression in normal keratinocytes and SCC cell lines correlates with Ki67 expression. A) Ki67, EGFR, cyclin-D1 and CCHCR1 gene expression profile of five normal epidermal KC and eight cutaneous SCC cell lines (heatmap). Signal values of the probe sets were compared to the mean signal values of each probe set in KCs. The colouring is based on the log2 values of the change in the signal values. The up-regulated genes are shown in red and down-regulated genes are shown in green. B) Correlation between CCHCR1 and Ki67 probe sets was calculated between the signal values of one CCHCR1 and one Ki67 probe set in the HG-U133 Plus 3.0 array. Pearson's correlation coefficient R = 0.88. C) CCHCR1 and D) Ki67 mRNA expression levels in the normal KCs and cutaneous SCC cell lines as measured by qRT-PCR (TaqMan). Expression levels of CCHCR1 and Ki67 in the normal KCs and SCC cell lines were analyzed by qRT-PCR and corrected for the β-actin mRNA levels in the same samples. E) Correlation between CCHCR1 and Ki67 probe sets was calculated between the signal values of CCHCR1 and Ki67 mRNA levels. Pearson's correlation coefficient R = 0.46.
    Figure Legend Snippet: CCHCR1 mRNA expression in normal keratinocytes and SCC cell lines correlates with Ki67 expression. A) Ki67, EGFR, cyclin-D1 and CCHCR1 gene expression profile of five normal epidermal KC and eight cutaneous SCC cell lines (heatmap). Signal values of the probe sets were compared to the mean signal values of each probe set in KCs. The colouring is based on the log2 values of the change in the signal values. The up-regulated genes are shown in red and down-regulated genes are shown in green. B) Correlation between CCHCR1 and Ki67 probe sets was calculated between the signal values of one CCHCR1 and one Ki67 probe set in the HG-U133 Plus 3.0 array. Pearson's correlation coefficient R = 0.88. C) CCHCR1 and D) Ki67 mRNA expression levels in the normal KCs and cutaneous SCC cell lines as measured by qRT-PCR (TaqMan). Expression levels of CCHCR1 and Ki67 in the normal KCs and SCC cell lines were analyzed by qRT-PCR and corrected for the β-actin mRNA levels in the same samples. E) Correlation between CCHCR1 and Ki67 probe sets was calculated between the signal values of CCHCR1 and Ki67 mRNA levels. Pearson's correlation coefficient R = 0.46.

    Techniques Used: Expressing, Quantitative RT-PCR

    CCHCR1, Ki67, and EGFR are coexpressed in grade I SCC and in normal skin. Serial sections of grade I SCC (A–C) and normal skin (H–J) were immunostained with antibodies against CCHCR1, Ki67, and EGFR. Higher magnifications of A–C are also shown (D–F, respectively). CCHCR1 staining (G) in an adjacent section to EGFR staining (F). CCHCR1 protein (A) is expressed in proliferative cancer cells at the invasive front of dermal cancer cell islands of an SCC in association with the hyperproliferation marker Ki67 (B) and EGFR (C). The Ki67 positive cells (E) express CCHCR1 (D). EGFR staining (F) associates with CCHCR1 staining (G) in adjacent sections. Normal skin samples express CCHCR1 (H) and EGFR (J) in basal KCs, while Ki67 expression (I) is more sparse. Arrows point at illustrative positions. Scale bars: (A–C) 50 µm; (D–G) 12.5 µm; (H–J) 25 µm.
    Figure Legend Snippet: CCHCR1, Ki67, and EGFR are coexpressed in grade I SCC and in normal skin. Serial sections of grade I SCC (A–C) and normal skin (H–J) were immunostained with antibodies against CCHCR1, Ki67, and EGFR. Higher magnifications of A–C are also shown (D–F, respectively). CCHCR1 staining (G) in an adjacent section to EGFR staining (F). CCHCR1 protein (A) is expressed in proliferative cancer cells at the invasive front of dermal cancer cell islands of an SCC in association with the hyperproliferation marker Ki67 (B) and EGFR (C). The Ki67 positive cells (E) express CCHCR1 (D). EGFR staining (F) associates with CCHCR1 staining (G) in adjacent sections. Normal skin samples express CCHCR1 (H) and EGFR (J) in basal KCs, while Ki67 expression (I) is more sparse. Arrows point at illustrative positions. Scale bars: (A–C) 50 µm; (D–G) 12.5 µm; (H–J) 25 µm.

    Techniques Used: Staining, Marker, Expressing

    16) Product Images from "Correlation of AR, EGFR, and HER2 Expression Levels in Prostate Cancer: Immunohistochemical Analysis and Chromogenic In Situ Hybridization"

    Article Title: Correlation of AR, EGFR, and HER2 Expression Levels in Prostate Cancer: Immunohistochemical Analysis and Chromogenic In Situ Hybridization

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    doi: 10.4143/crt.2012.44.1.50

    Epidermal growth factor recptor ( EGFR ) (A) and human epidermal growth factor recptor 2 ( HER2 ) (B) gene amplification examined by chromogenic in situ hybridization. One or 2 signals are visible in nuclei of adenocarcinoma cells. There is no evidence of gene amplification (CISH staining, ×400).
    Figure Legend Snippet: Epidermal growth factor recptor ( EGFR ) (A) and human epidermal growth factor recptor 2 ( HER2 ) (B) gene amplification examined by chromogenic in situ hybridization. One or 2 signals are visible in nuclei of adenocarcinoma cells. There is no evidence of gene amplification (CISH staining, ×400).

    Techniques Used: Amplification, Chromogenic In Situ Hybridization, Staining

    17) Product Images from "Breast Cancer Cell Invasion into a Three Dimensional Tumor-Stroma Microenvironment"

    Article Title: Breast Cancer Cell Invasion into a Three Dimensional Tumor-Stroma Microenvironment

    Journal: Scientific Reports

    doi: 10.1038/srep34094

    Investigation of EGFR and pEGFR. ( A ) Cells were stained for EGFR (red), pEGFR (green), and nuclei (blue) (scale bar: 20 μm). ( B ) Representative images of EGFR clusters with corresponding heat maps of relative intensities. ( C ) (+) EGF demonstrated significantly lower EGFR to cell area ratio. ( D , E ) (+) EGF displayed significantly higher pEGFR to cell area ratio and pEGFR to EGFR area ratio ( p
    Figure Legend Snippet: Investigation of EGFR and pEGFR. ( A ) Cells were stained for EGFR (red), pEGFR (green), and nuclei (blue) (scale bar: 20 μm). ( B ) Representative images of EGFR clusters with corresponding heat maps of relative intensities. ( C ) (+) EGF demonstrated significantly lower EGFR to cell area ratio. ( D , E ) (+) EGF displayed significantly higher pEGFR to cell area ratio and pEGFR to EGFR area ratio ( p

    Techniques Used: Staining

    18) Product Images from "Design and discovery of novel monastrol-1,3,5-triazines as potent anti-breast cancer agent via attenuating Epidermal Growth Factor Receptor tyrosine kinase"

    Article Title: Design and discovery of novel monastrol-1,3,5-triazines as potent anti-breast cancer agent via attenuating Epidermal Growth Factor Receptor tyrosine kinase

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05934-5

    Effect of compound 7l on the p-EGFR, EGFR, p-Akt and Akt as determined by western blot assay.
    Figure Legend Snippet: Effect of compound 7l on the p-EGFR, EGFR, p-Akt and Akt as determined by western blot assay.

    Techniques Used: Western Blot

    19) Product Images from "Appropriateness of Using Patient-Derived Xenograft Models for Pharmacologic Evaluation of Novel Therapies for Esophageal/Gastro-Esophageal Junction Cancers"

    Article Title: Appropriateness of Using Patient-Derived Xenograft Models for Pharmacologic Evaluation of Novel Therapies for Esophageal/Gastro-Esophageal Junction Cancers

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121872

    Selected molecular marker expression by immunohistochemistry (IHC). P53 in Line A and Ki-67 in Line H are examples of similar expression between patient, early passage (P1) and latest passage (P latest ) xenografts. P16 in Line H was selected to demonstrate the heterogeneity detected in the same tissue (P early showing both positive and negative expression). EGFR expression in Line E exhibited an increase in intensity from patient to xenografts while Her-2/ neu expression in Line A showed a decrease in intensity. These examples were included to demonstrate that the differences exhibited between patient tissue, early passage and latest passage xenografts were due to intrinsic heterogeneity and not to any specific patterns of expression.
    Figure Legend Snippet: Selected molecular marker expression by immunohistochemistry (IHC). P53 in Line A and Ki-67 in Line H are examples of similar expression between patient, early passage (P1) and latest passage (P latest ) xenografts. P16 in Line H was selected to demonstrate the heterogeneity detected in the same tissue (P early showing both positive and negative expression). EGFR expression in Line E exhibited an increase in intensity from patient to xenografts while Her-2/ neu expression in Line A showed a decrease in intensity. These examples were included to demonstrate that the differences exhibited between patient tissue, early passage and latest passage xenografts were due to intrinsic heterogeneity and not to any specific patterns of expression.

    Techniques Used: Marker, Expressing, Immunohistochemistry

    20) Product Images from "Spatial EGFR Dynamics and Metastatic Phenotypes Modulated by Upregulated EphB2 and Src Pathways in Advanced Prostate Cancer"

    Article Title: Spatial EGFR Dynamics and Metastatic Phenotypes Modulated by Upregulated EphB2 and Src Pathways in Advanced Prostate Cancer

    Journal: Cancers

    doi: 10.3390/cancers11121910

    Disruption of EphB2/Src pathways leads to attenuated cell motility, invasion, and EGFR diffusion in advanced prostate cancer cells. ( A ) Effective gene knockdowns in siRNA-treated DU145 and PC3. ( B ) Structured Illumination Microscopy (SIM) images of siRNA treated cells. Maximum intensity projection on the xy plane and orthogonal cross-sections (xz and yz) of DU145 and PC3 siRNA treated cells. ( C ) Quantification of cortical actin based on fluorescence intensities of xz and yz orthogonal projections along the apical plasma membrane. The number of projections analyzed is labeled on each bar. ( D ) EGFR diffusivities of the siRNAs treated cells. The error bar represents the standard error of the mean. ( E , F ) The image-based assays allow us to conduct the time-lapse analysis of cell migration and invasion on the siRNA-treated cells. The error bar represents the standard deviation. All statistical analyses were performed using the unpaired t -test. The asterisk represents the level of statistical significance for t -test: *** p
    Figure Legend Snippet: Disruption of EphB2/Src pathways leads to attenuated cell motility, invasion, and EGFR diffusion in advanced prostate cancer cells. ( A ) Effective gene knockdowns in siRNA-treated DU145 and PC3. ( B ) Structured Illumination Microscopy (SIM) images of siRNA treated cells. Maximum intensity projection on the xy plane and orthogonal cross-sections (xz and yz) of DU145 and PC3 siRNA treated cells. ( C ) Quantification of cortical actin based on fluorescence intensities of xz and yz orthogonal projections along the apical plasma membrane. The number of projections analyzed is labeled on each bar. ( D ) EGFR diffusivities of the siRNAs treated cells. The error bar represents the standard error of the mean. ( E , F ) The image-based assays allow us to conduct the time-lapse analysis of cell migration and invasion on the siRNA-treated cells. The error bar represents the standard deviation. All statistical analyses were performed using the unpaired t -test. The asterisk represents the level of statistical significance for t -test: *** p

    Techniques Used: Diffusion-based Assay, Microscopy, Fluorescence, Labeling, Migration, Standard Deviation

    Dasatinib inhibits proliferation, migration, invasion, and EGFR diffusivity in advanced prostate cancer cells. ( A ) Src is highly upregulated in PC3 (2.5x) and DU145 (1.6x) as compared to LNCaP shown in Western blots. EphB2 is overexpressed in both PC3 and DU145. Both proteins are almost not expressed in LNCaP-Abl. ( B ) Src is present in these cell lines. There is an intense level of Src on the PC3 cell membrane. ( C ) Immunostaining of EphB2 protein is present in plasma and membrane. ( D ) Image-based IncuCyte assays allow us to conduct the time-lapse analysis of cell proliferation, migration, and invasion of the cells treated with or without dasatinib. The dasatinib significantly inhibits the proliferation, migration, and invasion of DU145 and PC3 cells but reduces the EGFR diffusivity in only PC3 cells. The mean value of each bar was measured at the end time of each assay or at the 48th hour. All statistical analyses were performed using the unpaired t -test. The asterisk represents the level of statistical significance for t -test: *** p
    Figure Legend Snippet: Dasatinib inhibits proliferation, migration, invasion, and EGFR diffusivity in advanced prostate cancer cells. ( A ) Src is highly upregulated in PC3 (2.5x) and DU145 (1.6x) as compared to LNCaP shown in Western blots. EphB2 is overexpressed in both PC3 and DU145. Both proteins are almost not expressed in LNCaP-Abl. ( B ) Src is present in these cell lines. There is an intense level of Src on the PC3 cell membrane. ( C ) Immunostaining of EphB2 protein is present in plasma and membrane. ( D ) Image-based IncuCyte assays allow us to conduct the time-lapse analysis of cell proliferation, migration, and invasion of the cells treated with or without dasatinib. The dasatinib significantly inhibits the proliferation, migration, and invasion of DU145 and PC3 cells but reduces the EGFR diffusivity in only PC3 cells. The mean value of each bar was measured at the end time of each assay or at the 48th hour. All statistical analyses were performed using the unpaired t -test. The asterisk represents the level of statistical significance for t -test: *** p

    Techniques Used: Migration, Western Blot, Immunostaining

    21) Product Images from "Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging"

    Article Title: Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging

    Journal: Science Advances

    doi: 10.1126/sciadv.1600265

    Schematic presentation of the FRET imaging approaches used in this study. (1) Extracellular FRET between Tb- and QD-functionalized antibodies that bind to different epitopes of EGFR on the cell membrane. (2) Intracellular (cytosol) FRET from Tb-to-QD and FRET relays from Tb-to-QD-to-dye using microinjected QDs conjugated with Alexa Fluor 647 (AF) and Lumi4-Tb peptides via hexahistidine (His 6 ) self-assembly. (3) Intracellular (endosomes/lysosomes) FRET from Tb-to-QD using CPP-mediated endocytosis of QDs conjugated with Lumi4-Tb peptides via hexahistidine self-assembly.
    Figure Legend Snippet: Schematic presentation of the FRET imaging approaches used in this study. (1) Extracellular FRET between Tb- and QD-functionalized antibodies that bind to different epitopes of EGFR on the cell membrane. (2) Intracellular (cytosol) FRET from Tb-to-QD and FRET relays from Tb-to-QD-to-dye using microinjected QDs conjugated with Alexa Fluor 647 (AF) and Lumi4-Tb peptides via hexahistidine (His 6 ) self-assembly. (3) Intracellular (endosomes/lysosomes) FRET from Tb-to-QD using CPP-mediated endocytosis of QDs conjugated with Lumi4-Tb peptides via hexahistidine self-assembly.

    Techniques Used: Imaging, Conditioned Place Preference

    Extracellular Tb-to-QD FRET using immunostaining of EGFR. ( A and B ) Tb-to-QD FRET detected on A431 cells with QD- and Tb-conjugated antibodies (A) and nanobodies (B). For both immunostaining approaches, the time-gated Tb (TG Tb PL) and QD (TG QD PL) channels reveal bright PL signals originating mainly from the cell membranes. ( C and D ) In contrast, staining with only QD-antibodies (C) or only Tb-antibodies (D) does not result in TG PL in the QD channel (TG QD PL, right), and only the pure QD SS PL (C, SS QD PL, left) or the pure TG Tb PL (D, left) become visible. Excitation and emission wavelengths for the different detection channels were as follows: λ ex = 365 nm and λ em = 494 ± 10 nm for TG Tb PL, λ ex = 365 nm and λ em = 655 ± 20 nm for TG QD PL, and λ ex = 545 ± 15 nm and λ em = 610 ± 35 nm for SS QD PL. For TG images, the number of integrations was 220 and 110 for the TG Tb PL and TG QD PL channels, respectively. Tb-to-QD FRET channel images (TG QD PL) were corrected for spectral crosstalk, and each TG QD PL channel image in this figure is presented at identical contrast. Scale bars, 20 μm.
    Figure Legend Snippet: Extracellular Tb-to-QD FRET using immunostaining of EGFR. ( A and B ) Tb-to-QD FRET detected on A431 cells with QD- and Tb-conjugated antibodies (A) and nanobodies (B). For both immunostaining approaches, the time-gated Tb (TG Tb PL) and QD (TG QD PL) channels reveal bright PL signals originating mainly from the cell membranes. ( C and D ) In contrast, staining with only QD-antibodies (C) or only Tb-antibodies (D) does not result in TG PL in the QD channel (TG QD PL, right), and only the pure QD SS PL (C, SS QD PL, left) or the pure TG Tb PL (D, left) become visible. Excitation and emission wavelengths for the different detection channels were as follows: λ ex = 365 nm and λ em = 494 ± 10 nm for TG Tb PL, λ ex = 365 nm and λ em = 655 ± 20 nm for TG QD PL, and λ ex = 545 ± 15 nm and λ em = 610 ± 35 nm for SS QD PL. For TG images, the number of integrations was 220 and 110 for the TG Tb PL and TG QD PL channels, respectively. Tb-to-QD FRET channel images (TG QD PL) were corrected for spectral crosstalk, and each TG QD PL channel image in this figure is presented at identical contrast. Scale bars, 20 μm.

    Techniques Used: Immunostaining, Staining

    22) Product Images from "YL143, a novel mutant selective irreversible EGFR inhibitor, overcomes EGFRL858R, T790M‐mutant resistance in vitro and in vivo"

    Article Title: YL143, a novel mutant selective irreversible EGFR inhibitor, overcomes EGFRL858R, T790M‐mutant resistance in vitro and in vivo

    Journal: Cancer Medicine

    doi: 10.1002/cam4.1392

    YL143 suppresses EGFR signaling pathway in H1975 cells (A) YL143 potently inhibits the activation of EGFR signals in H1975 NSCLC. Cells were treated with or without compound YL143 for 4 h at indicated concentration, respectively. Cells were harvested for Western blot analysis. (B) Wash‐out assay demonstrates the irreversible binding of YL143 with EGFR L858R/T790M. NCI‐H1975 cells were treated with or without compound YL143(100 nmol/L) for 4 h, and then the medium with compound was removed and fresh medium was added. At the indicated time points, cells were harvest and proteins were extracted and subjected to Western blot analysis.
    Figure Legend Snippet: YL143 suppresses EGFR signaling pathway in H1975 cells (A) YL143 potently inhibits the activation of EGFR signals in H1975 NSCLC. Cells were treated with or without compound YL143 for 4 h at indicated concentration, respectively. Cells were harvested for Western blot analysis. (B) Wash‐out assay demonstrates the irreversible binding of YL143 with EGFR L858R/T790M. NCI‐H1975 cells were treated with or without compound YL143(100 nmol/L) for 4 h, and then the medium with compound was removed and fresh medium was added. At the indicated time points, cells were harvest and proteins were extracted and subjected to Western blot analysis.

    Techniques Used: Activation Assay, Concentration Assay, Western Blot, Binding Assay

    YL143 is a novel irreversible EGFR inhibitor. (A) Structure of YL143 and XTF262. (B) The binding mode of YL143 with EGFR predicted by the molecular docking simulations. (C) Kinase inhibitory activity of YL143 on EGFRWT, EGFRL858R, EGFRT790M and EGFRL858R/T790M by FRET‐based Z′‐Lyte assay.
    Figure Legend Snippet: YL143 is a novel irreversible EGFR inhibitor. (A) Structure of YL143 and XTF262. (B) The binding mode of YL143 with EGFR predicted by the molecular docking simulations. (C) Kinase inhibitory activity of YL143 on EGFRWT, EGFRL858R, EGFRT790M and EGFRL858R/T790M by FRET‐based Z′‐Lyte assay.

    Techniques Used: Binding Assay, Activity Assay

    23) Product Images from "Enhanced expression of epidermal growth factor receptor gene in gastric mucosal cells by the serum derived from rats treated with electroacupuncture at stomach meridian acupoints"

    Article Title: Enhanced expression of epidermal growth factor receptor gene in gastric mucosal cells by the serum derived from rats treated with electroacupuncture at stomach meridian acupoints

    Journal:

    doi: 10.3748/wjg.v12.i34.5557

    Electrophoresis of EGFR mRNA and GAPDH mRNA RT-PCR product in gastric mucosal cells. M: Marker; A: Normal group; B: Model group; C: Model serum group; D: Stomach serum group; E: Gallbladder serum group.
    Figure Legend Snippet: Electrophoresis of EGFR mRNA and GAPDH mRNA RT-PCR product in gastric mucosal cells. M: Marker; A: Normal group; B: Model group; C: Model serum group; D: Stomach serum group; E: Gallbladder serum group.

    Techniques Used: Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Marker

    24) Product Images from "EGFR and C/EBP-β oncogenic signaling is bidirectional in human glioma and varies with the C/EBP-β isoform"

    Article Title: EGFR and C/EBP-β oncogenic signaling is bidirectional in human glioma and varies with the C/EBP-β isoform

    Journal: The FASEB Journal

    doi: 10.1096/fj.201600550R

    EGFR overexpression and activation causes nuclear translocation of C/EBP-β. A ) Western blot for nuclear (N) and cytoplasmic (C) extracts showing differential expression of C/EBP-β (LAP1/2) in U87MG and U87MG-WT cells in the presence and absence of EGF. Expression of C/EBP-β is shifted from the cytoplasmic compartment to the nuclear compartment in the presence of EGF in U87MG and U87MG-WT. Topoisomerase I and actin were loading controls for nuclear and cytoplasmic lysate, respectively . B ) Western blot for nuclear and cytoplasmic extracts showing the increased detection of phosphorylated (activated) EGFR (Y1068) in the presence of EGF. Activation of EGFR by EGF also leads to reduced expression of EGFR . C ) Immunofluorescence of U87MG demonstrating increased nuclear expression of C/EBP-β in presence of EGF and its attenuation by AG1478 (EGF+AG1478), an inhibitor of EGFR phosphorylation . D ) EMSA demonstrated increased binding of C/EBP-β on its cognate DNA (arrow) in U87MG-WT compared to U87MG and also shows the increased binding of C/EBP-β in the presence of EGF in both cell lines. Densitometry values for intensity of bands are noted at the bottom of each lane. Asterisk indicates nonspecific and free probes.
    Figure Legend Snippet: EGFR overexpression and activation causes nuclear translocation of C/EBP-β. A ) Western blot for nuclear (N) and cytoplasmic (C) extracts showing differential expression of C/EBP-β (LAP1/2) in U87MG and U87MG-WT cells in the presence and absence of EGF. Expression of C/EBP-β is shifted from the cytoplasmic compartment to the nuclear compartment in the presence of EGF in U87MG and U87MG-WT. Topoisomerase I and actin were loading controls for nuclear and cytoplasmic lysate, respectively . B ) Western blot for nuclear and cytoplasmic extracts showing the increased detection of phosphorylated (activated) EGFR (Y1068) in the presence of EGF. Activation of EGFR by EGF also leads to reduced expression of EGFR . C ) Immunofluorescence of U87MG demonstrating increased nuclear expression of C/EBP-β in presence of EGF and its attenuation by AG1478 (EGF+AG1478), an inhibitor of EGFR phosphorylation . D ) EMSA demonstrated increased binding of C/EBP-β on its cognate DNA (arrow) in U87MG-WT compared to U87MG and also shows the increased binding of C/EBP-β in the presence of EGF in both cell lines. Densitometry values for intensity of bands are noted at the bottom of each lane. Asterisk indicates nonspecific and free probes.

    Techniques Used: Over Expression, Activation Assay, Translocation Assay, Western Blot, Expressing, Immunofluorescence, Binding Assay

    EGFR and C/EBP-β regulate each other at mRNA level. A ) qRT-PCR showing increased mRNA levels of EGFR and C/EBP-b relative to GAPDH in U87MG-WT vs. U87MG cells. All the experiments were performed with 3 technical and 3 biologic replicates. * P
    Figure Legend Snippet: EGFR and C/EBP-β regulate each other at mRNA level. A ) qRT-PCR showing increased mRNA levels of EGFR and C/EBP-b relative to GAPDH in U87MG-WT vs. U87MG cells. All the experiments were performed with 3 technical and 3 biologic replicates. * P

    Techniques Used: Quantitative RT-PCR

    25) Product Images from "Human cancers converge at the HIF-2? oncogenic axis"

    Article Title: Human cancers converge at the HIF-2? oncogenic axis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0906432106

    HIF-2α activates the EGFR and IGF1R tyrosine kinases and their downstream signaling molecules. ( A ) Relative levels of RTK phosphorylation in serum-starved human cancer cell lines stably expressing scramble control, HIF-1α or HIF-2α
    Figure Legend Snippet: HIF-2α activates the EGFR and IGF1R tyrosine kinases and their downstream signaling molecules. ( A ) Relative levels of RTK phosphorylation in serum-starved human cancer cell lines stably expressing scramble control, HIF-1α or HIF-2α

    Techniques Used: Stable Transfection, Expressing

    26) Product Images from "Targeting Receptor Tyrosine Kinase on Lymphatic Endothelial Cells for the Therapy of Colon Cancer Lymph Node Metastasis 1"

    Article Title: Targeting Receptor Tyrosine Kinase on Lymphatic Endothelial Cells for the Therapy of Colon Cancer Lymph Node Metastasis 1

    Journal: Neoplasia (New York, N.Y.)

    doi:

    Double-immunofluorescence staining for total andphosphorylated VEGFR-2, VEGFR-3, and EGFR in peritumoral lymphatic vessels. (A) Samples were stained with anti-LYVE-1 antibody (green) and anti-EGFR, anti-VEGFR-2, or anti-VEGFR-3 (red) antibody, as described in the Materials and Methods section. The colocalization of LYVE-1 and receptors appears as yellow fluorescence. The expression of EGFR, VEGFR-2, and VEGFR-3 was found in peritumoral lymphatic vessels of mice treated with control or AEE788. (B) Phosphorylation of EGFR and VEGFR-2/3 in lymphatic endothelial cells was decreased by treatment with AEE788. Scale bars = 100 µm.
    Figure Legend Snippet: Double-immunofluorescence staining for total andphosphorylated VEGFR-2, VEGFR-3, and EGFR in peritumoral lymphatic vessels. (A) Samples were stained with anti-LYVE-1 antibody (green) and anti-EGFR, anti-VEGFR-2, or anti-VEGFR-3 (red) antibody, as described in the Materials and Methods section. The colocalization of LYVE-1 and receptors appears as yellow fluorescence. The expression of EGFR, VEGFR-2, and VEGFR-3 was found in peritumoral lymphatic vessels of mice treated with control or AEE788. (B) Phosphorylation of EGFR and VEGFR-2/3 in lymphatic endothelial cells was decreased by treatment with AEE788. Scale bars = 100 µm.

    Techniques Used: Double Immunofluorescence Staining, Staining, Fluorescence, Expressing, Mouse Assay

    27) Product Images from "The ubiquitin-specific protease USP2a enhances tumor progression by targeting cyclin A1 in bladder cancer"

    Article Title: The ubiquitin-specific protease USP2a enhances tumor progression by targeting cyclin A1 in bladder cancer

    Journal: Cell Cycle

    doi: 10.4161/cc.11.6.19550

    USP2a stabilized cyclin A1. (A) USP2a WT expressing T24 cells expressed higher level of cyclin D1 and cyclin A1. (B) USP2a-targeted oligos, siUSP2a, reversed an increase of cyclin D1 and cyclin A1. (C) Physical interaction of USP2a and cyclin A1. Whole-cell lysates (200 µg) from T24 cells were applied to immunoprecipitation (IP) with anti-cyclin A1 antibody, and IP product was blotted with anti-USP2a antibody (∼70 kDa). The reciprocal co-immunoprecipitation assay was not applied due to no difference between cyclin A1 and IgG (50–55 kDa), although the IP itself was working and we could detect EGFR and FASN co-immunoprecipitated with USP2a. (D) Cyclin A1 degradation was measured in absence or presence of 10 µg/ml CHX in the T24-USP2a WT and T24-Vec cells.
    Figure Legend Snippet: USP2a stabilized cyclin A1. (A) USP2a WT expressing T24 cells expressed higher level of cyclin D1 and cyclin A1. (B) USP2a-targeted oligos, siUSP2a, reversed an increase of cyclin D1 and cyclin A1. (C) Physical interaction of USP2a and cyclin A1. Whole-cell lysates (200 µg) from T24 cells were applied to immunoprecipitation (IP) with anti-cyclin A1 antibody, and IP product was blotted with anti-USP2a antibody (∼70 kDa). The reciprocal co-immunoprecipitation assay was not applied due to no difference between cyclin A1 and IgG (50–55 kDa), although the IP itself was working and we could detect EGFR and FASN co-immunoprecipitated with USP2a. (D) Cyclin A1 degradation was measured in absence or presence of 10 µg/ml CHX in the T24-USP2a WT and T24-Vec cells.

    Techniques Used: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay

    28) Product Images from "Interleukin-13 receptor alpha 2 cooperates with EGFRvIII signaling to promote glioblastoma multiforme"

    Article Title: Interleukin-13 receptor alpha 2 cooperates with EGFRvIII signaling to promote glioblastoma multiforme

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01392-9

    Deletion of the cytoplasmic domain of IL-13Rα2 resulted in a loss of physical interaction with EGFRvIII and enhanced proliferation is abolished. a Whole-cell lysates prepared from stable cell line Gli36.IL-13Rα2/EGFRvIII cells were used for immunoprecipitation with anti-IL-13Rα2 antibody, then immunoprobed with an anti-EGFR antibody. IgG served as control while unprecipitated extracts serve as input. b Similar cell lysates were reverse immunoprecipitated with anti-EGFR antibody, then immunoprobed with an anti-IL13Rα2antibody. Lysates from Gli36.EGFRvIII served as additional control c Gli36.IL-13Rα2/EGFRvIII cell lysates were immunoprecipitated with anti-EGFR antibody, then immunoprobed with anti-Grb antibody. To further examine the domains of interaction, IL-13Rα2 and EGFR mutants were used. Gli36.EGFRvIII cells were first transfected with pIRESneo2 (Vector), IL-13Rα2 full length (Wild-type) and IL-13Rα2 Cyt tail deleted constructs (Mutant) and then analyzed by d cell proliferation assay at the indicated time points, f co-immunoprecipitation, and h PLA assays. Findings were validated using Gli36.IL-13Rα2 cells transiently transfected with vector (CTRL), full length/wild-type EGFRvIII, DK, and DY3 mutants. e proliferation outputs, g co-immunoprecipitation, i and PLA assay were performed. j represent the corresponding positive and negative controls
    Figure Legend Snippet: Deletion of the cytoplasmic domain of IL-13Rα2 resulted in a loss of physical interaction with EGFRvIII and enhanced proliferation is abolished. a Whole-cell lysates prepared from stable cell line Gli36.IL-13Rα2/EGFRvIII cells were used for immunoprecipitation with anti-IL-13Rα2 antibody, then immunoprobed with an anti-EGFR antibody. IgG served as control while unprecipitated extracts serve as input. b Similar cell lysates were reverse immunoprecipitated with anti-EGFR antibody, then immunoprobed with an anti-IL13Rα2antibody. Lysates from Gli36.EGFRvIII served as additional control c Gli36.IL-13Rα2/EGFRvIII cell lysates were immunoprecipitated with anti-EGFR antibody, then immunoprobed with anti-Grb antibody. To further examine the domains of interaction, IL-13Rα2 and EGFR mutants were used. Gli36.EGFRvIII cells were first transfected with pIRESneo2 (Vector), IL-13Rα2 full length (Wild-type) and IL-13Rα2 Cyt tail deleted constructs (Mutant) and then analyzed by d cell proliferation assay at the indicated time points, f co-immunoprecipitation, and h PLA assays. Findings were validated using Gli36.IL-13Rα2 cells transiently transfected with vector (CTRL), full length/wild-type EGFRvIII, DK, and DY3 mutants. e proliferation outputs, g co-immunoprecipitation, i and PLA assay were performed. j represent the corresponding positive and negative controls

    Techniques Used: Stable Transfection, Immunoprecipitation, Transfection, Plasmid Preparation, Construct, Mutagenesis, Proliferation Assay, Proximity Ligation Assay

    Enhanced cellular proliferation mediated by IL-13Rα2 is specific to EGFRvIII, and not WT EGFR. a U251-E6 or c U251-E18 cells were treated with or without tetracycline (Tet). At indicated time points, immunoblot analysis was carried out. Gli36, Gli36.EGFRvIII cell lysates were included as negative or positive controls for EGFRvIII, respectively. Growth kinetics of b U251-E6 and d U251-E18 was determined by CCK-8 assay. Percent cell viability was normalized to day 1 (without induction). All data are represented as mean ± SEM. Unpaired t -test *** p
    Figure Legend Snippet: Enhanced cellular proliferation mediated by IL-13Rα2 is specific to EGFRvIII, and not WT EGFR. a U251-E6 or c U251-E18 cells were treated with or without tetracycline (Tet). At indicated time points, immunoblot analysis was carried out. Gli36, Gli36.EGFRvIII cell lysates were included as negative or positive controls for EGFRvIII, respectively. Growth kinetics of b U251-E6 and d U251-E18 was determined by CCK-8 assay. Percent cell viability was normalized to day 1 (without induction). All data are represented as mean ± SEM. Unpaired t -test *** p

    Techniques Used: CCK-8 Assay

    IL-13Rα2 mediate greater tumorigenic potential with EGFRvIII, and not WT EGFR. a Tumor volume b and tumor weight of tetracycline regulatable U251 gliomas (U251-E6 and U251-E18 was examined in vivo. Bars depict the mean values and error bars represent 95% confidence intervals. P -values were calculated using ANOVA with Tukey’s multiple comparison test * p
    Figure Legend Snippet: IL-13Rα2 mediate greater tumorigenic potential with EGFRvIII, and not WT EGFR. a Tumor volume b and tumor weight of tetracycline regulatable U251 gliomas (U251-E6 and U251-E18 was examined in vivo. Bars depict the mean values and error bars represent 95% confidence intervals. P -values were calculated using ANOVA with Tukey’s multiple comparison test * p

    Techniques Used: In Vivo

    GBM patients co-expressing EGFR and IL-13Rα2 correlate to poor survival where the overexpression of IL-13Rα2 alone leads to enhance cell migration but not proliferation. Kaplan−Meier survival analysis of a all gliomas patients; b GBM patients from REMBRANDT database from National Cancer Institute (USA). Patients overexpressing EGFR mRNA by 2-fold (blue) with high (red), intermediate (yellow) and low (green) levels of IL-13Rα2 expression were shown. The log-rank p -values were indicated. c Kaplan−Meier survival plots for patients expressing high YKL-40 mRNA levels TCGA. High IL-13Rα2 expression group (red) and low IL-13Rα2 expression group (blue) were determined by aggregating all patients whose z -score normalized expression was above or below 0, respectively (Log-rank test p -value = 0.0374). Immunoblotting analysis showed the expression of EGFR and IL-13Rα2 protein levels were determined from d a panel of 10 patient-derived GBM e and the isogenic cell lines generated from Gli36 glioma cells. Pan-actin or β tubulin served as internal loading controls. f Cell proliferation and g Cell cycle analysis were performed with Gli36 and Gli36.IL-13Rα2 cells h Soft agar colony formation assay was performed, Gli36.EGFRvIII was used as a positive control. i In vitro migration and j invasion assays were determined in Gli36 and Gli36.IL-13Rα2 cells. All data are represented as mean ± SEM, unpaired t -test ** p
    Figure Legend Snippet: GBM patients co-expressing EGFR and IL-13Rα2 correlate to poor survival where the overexpression of IL-13Rα2 alone leads to enhance cell migration but not proliferation. Kaplan−Meier survival analysis of a all gliomas patients; b GBM patients from REMBRANDT database from National Cancer Institute (USA). Patients overexpressing EGFR mRNA by 2-fold (blue) with high (red), intermediate (yellow) and low (green) levels of IL-13Rα2 expression were shown. The log-rank p -values were indicated. c Kaplan−Meier survival plots for patients expressing high YKL-40 mRNA levels TCGA. High IL-13Rα2 expression group (red) and low IL-13Rα2 expression group (blue) were determined by aggregating all patients whose z -score normalized expression was above or below 0, respectively (Log-rank test p -value = 0.0374). Immunoblotting analysis showed the expression of EGFR and IL-13Rα2 protein levels were determined from d a panel of 10 patient-derived GBM e and the isogenic cell lines generated from Gli36 glioma cells. Pan-actin or β tubulin served as internal loading controls. f Cell proliferation and g Cell cycle analysis were performed with Gli36 and Gli36.IL-13Rα2 cells h Soft agar colony formation assay was performed, Gli36.EGFRvIII was used as a positive control. i In vitro migration and j invasion assays were determined in Gli36 and Gli36.IL-13Rα2 cells. All data are represented as mean ± SEM, unpaired t -test ** p

    Techniques Used: Expressing, Over Expression, Migration, Derivative Assay, Generated, Cell Cycle Assay, Soft Agar Assay, Positive Control, In Vitro

    29) Product Images from "18F-fludrodeoxyglucose maximal standardized uptake value and metabolic tumor burden are associated with major chemotherapy-related tumor markers in NSCLC patients"

    Article Title: 18F-fludrodeoxyglucose maximal standardized uptake value and metabolic tumor burden are associated with major chemotherapy-related tumor markers in NSCLC patients

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S113832

    Correlationship analysis among the parameters. Notes: ( A ) SUV max was significantly correlated with p53 IHC score ( R =0.473, P =0.001); ( B ) MTV was significantly correlated with ERCC1 IHC score ( R =0.667, P =0.000); ( C ) TLG was significantly correlated with ERCC1 IHC score ( R =0.712, P =0.000). Abbreviations: EGFR, epidermal growth factor receptor; ERCC1, excision repair cross-complementing group 1 protein; IHC, immunohistochemistry; MTV, metabolic tumor volume; SUV max , maximum standardized uptake value; TLG, total lesion glycolysis.
    Figure Legend Snippet: Correlationship analysis among the parameters. Notes: ( A ) SUV max was significantly correlated with p53 IHC score ( R =0.473, P =0.001); ( B ) MTV was significantly correlated with ERCC1 IHC score ( R =0.667, P =0.000); ( C ) TLG was significantly correlated with ERCC1 IHC score ( R =0.712, P =0.000). Abbreviations: EGFR, epidermal growth factor receptor; ERCC1, excision repair cross-complementing group 1 protein; IHC, immunohistochemistry; MTV, metabolic tumor volume; SUV max , maximum standardized uptake value; TLG, total lesion glycolysis.

    Techniques Used: Immunohistochemistry

    Representative images of immunohistochemistry. Notes: ( A ) EGFR, ( B ) p53, and ( C ) ERCC1; (magnification, ×400). Abbreviations: EGFR, epidermal growth factor receptor; ERCC1, excision repair cross-complementing group 1 protein.
    Figure Legend Snippet: Representative images of immunohistochemistry. Notes: ( A ) EGFR, ( B ) p53, and ( C ) ERCC1; (magnification, ×400). Abbreviations: EGFR, epidermal growth factor receptor; ERCC1, excision repair cross-complementing group 1 protein.

    Techniques Used: Immunohistochemistry

    The ROC curve. Notes: ( A ) ROC curve for the optimal cutoff value of SUV max suggesting p53-positive NSCLC. Area under the curve: 0.737; 95% CI: 0.592–0.881; P =0.006. A SUV max value of 7.68 or higher suggests a NSCLC to be p53 positive with a sensitivity of 91% and specificity of 50%; ( B ) ROC curve for the optimal cutoff value of MTV suggesting ERCC1-positive NSCLC. Area under the curve: 0.825; 95% CI: 0.705–0.945; P =0.000. A MTV value of 23.62 cm 3 or lower suggests NSCLC to be ERCC1 positive with a sensitivity of 83% and specificity of 69%; ( C ) ROC curve for the optimal cutoff value of TLG suggesting ERCC1-positive NSCLC. Area under the curve: 0.835; 95% CI: 0.714–0.956; P =0.000. A TLG value of 129.65 or lower suggests a NSCLC to be p53 positive, with a sensitivity of 80% and specificity of 75%. Abbreviations: CI, confidence interval; EGFR, epidermal growth factor receptor; ERCC1, excision repair cross-complementing group 1 protein; MTV, metabolic tumor volume; NSCLC, non-small cell lung cancer; ROC, receiver operating characteristics; SUV max , maximum standardized uptake value; TLG, total lesion glycolysis.
    Figure Legend Snippet: The ROC curve. Notes: ( A ) ROC curve for the optimal cutoff value of SUV max suggesting p53-positive NSCLC. Area under the curve: 0.737; 95% CI: 0.592–0.881; P =0.006. A SUV max value of 7.68 or higher suggests a NSCLC to be p53 positive with a sensitivity of 91% and specificity of 50%; ( B ) ROC curve for the optimal cutoff value of MTV suggesting ERCC1-positive NSCLC. Area under the curve: 0.825; 95% CI: 0.705–0.945; P =0.000. A MTV value of 23.62 cm 3 or lower suggests NSCLC to be ERCC1 positive with a sensitivity of 83% and specificity of 69%; ( C ) ROC curve for the optimal cutoff value of TLG suggesting ERCC1-positive NSCLC. Area under the curve: 0.835; 95% CI: 0.714–0.956; P =0.000. A TLG value of 129.65 or lower suggests a NSCLC to be p53 positive, with a sensitivity of 80% and specificity of 75%. Abbreviations: CI, confidence interval; EGFR, epidermal growth factor receptor; ERCC1, excision repair cross-complementing group 1 protein; MTV, metabolic tumor volume; NSCLC, non-small cell lung cancer; ROC, receiver operating characteristics; SUV max , maximum standardized uptake value; TLG, total lesion glycolysis.

    Techniques Used:

    Expressions of SUV max , MTV, and TLG and three different biomarkers. Notes: The differences of SUV max ( A ), MTV ( B ), and TLG ( C ) in the expressions of EGFR, p53, and ERCC1 among NSCLC patients. * P
    Figure Legend Snippet: Expressions of SUV max , MTV, and TLG and three different biomarkers. Notes: The differences of SUV max ( A ), MTV ( B ), and TLG ( C ) in the expressions of EGFR, p53, and ERCC1 among NSCLC patients. * P

    Techniques Used:

    30) Product Images from "EpCAM ectodomain EpEX is a ligand of EGFR that counteracts EGF-mediated epithelial-mesenchymal transition through modulation of phospho-ERK1/2 in head and neck cancers"

    Article Title: EpCAM ectodomain EpEX is a ligand of EGFR that counteracts EGF-mediated epithelial-mesenchymal transition through modulation of phospho-ERK1/2 in head and neck cancers

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2006624

    EGFR and EpCAM levels are molecular determinants of EMT induction, ERK activation, and migration. (A) Kyse30 cells were transfected with EGFR-specific siRNAs (pool of n ], control shRNA (shRNA ctrl), and a combination of EGFR siRNA and EpCAM shRNA (double KD). Expression of EGFR and EpCAM was assessed by immunoblotting with specific antibodies. Equal loading was confirmed by detecting actin levels. Clinical quadrants’ equivalents are indicated. Shown are representative results from n = 3 independent experiments. (B) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGF low (1.8 nM) and EGF high (9 nM). Cell morphology was assessed after 72 hr. Shown are representative images from n = 3 independent experiments. (C) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGF low (1.8 nM) for the indicated time points, and ERK1/2 phosphorylation was assessed by immunoblotting. Levels of ERK1/2 and actin were assessed in parallel. Shown are representative results from n = 3 independent experiments. (D) Quadrant 1 to 4 equivalents of Kyse30 cell variants were either kept untreated (control) or were treated with EGF low (1.8 nM). After 72 hr, mRNA transcript levels of Slug were assessed by qRT-PCR with specific primers. Shown are means with SDs from n . (E) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGF high (9 nM) and subjected to a scratch assay. Relative migration was quantified from representative micrographs of each cell line. Shown are means with SDs from n . EGF, epidermal growth factor; EGFR, EGF receptor; EpCAM, epithelial cell adhesion molecule; EMT, epithelial-mesenchymal transition; ERK, extracellular signal–regulated kinase; KD, knockdown; pERK1/2, phosphorylated ERK1/2; qRT-PCR, quantitative real-time PCR; SD, standard deviation; shRNA, short hairpin RNA; siRNA, small interfering RNA; WT, wild type.
    Figure Legend Snippet: EGFR and EpCAM levels are molecular determinants of EMT induction, ERK activation, and migration. (A) Kyse30 cells were transfected with EGFR-specific siRNAs (pool of n ], control shRNA (shRNA ctrl), and a combination of EGFR siRNA and EpCAM shRNA (double KD). Expression of EGFR and EpCAM was assessed by immunoblotting with specific antibodies. Equal loading was confirmed by detecting actin levels. Clinical quadrants’ equivalents are indicated. Shown are representative results from n = 3 independent experiments. (B) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGF low (1.8 nM) and EGF high (9 nM). Cell morphology was assessed after 72 hr. Shown are representative images from n = 3 independent experiments. (C) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGF low (1.8 nM) for the indicated time points, and ERK1/2 phosphorylation was assessed by immunoblotting. Levels of ERK1/2 and actin were assessed in parallel. Shown are representative results from n = 3 independent experiments. (D) Quadrant 1 to 4 equivalents of Kyse30 cell variants were either kept untreated (control) or were treated with EGF low (1.8 nM). After 72 hr, mRNA transcript levels of Slug were assessed by qRT-PCR with specific primers. Shown are means with SDs from n . (E) Quadrant 1 to 4 equivalents of Kyse30 cell variants were treated with EGF high (9 nM) and subjected to a scratch assay. Relative migration was quantified from representative micrographs of each cell line. Shown are means with SDs from n . EGF, epidermal growth factor; EGFR, EGF receptor; EpCAM, epithelial cell adhesion molecule; EMT, epithelial-mesenchymal transition; ERK, extracellular signal–regulated kinase; KD, knockdown; pERK1/2, phosphorylated ERK1/2; qRT-PCR, quantitative real-time PCR; SD, standard deviation; shRNA, short hairpin RNA; siRNA, small interfering RNA; WT, wild type.

    Techniques Used: Activation Assay, Migration, Transfection, shRNA, Expressing, Quantitative RT-PCR, Wound Healing Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Small Interfering RNA

    EGFR and EpCAM expression predicts differential clinical outcome of HNSCCs. (A, C) Expression of EGFR and EpCAM was assessed in serial cryosections of normal mucosa and primary HNSCCs by IHC staining. Shown are representative examples of EGFR high /EpCAM high (A), EGFR high /EpCAM low , and EGFR low /EpCAM high (C) tumors at 100×, 200×, and 400× magnifications. (B) OS probability of HNSCC patients from the LMU cohort ( n = 180) and from a subcohort of the HNSCC TCGA cohort ( n ]. Protein expression data for EGFR from the RPPA data were used for the TCGA cohort. OS is represented as Kaplan-Meier survival curves with p . (D, F) IHC scores of EGFR and EpCAM expression were assessed in n = 180 primary HNSCCs of the LMU cohort (D) and in n = 87 HPV-negative primary HNSCCs of the LMU cohort (F). Expression correlation of EGFR and EpCAM is plotted and subdivided according to a cutoff threshold of 150 (score range 0–300). Numbers and percentages of patients within subgroups are indicated in each quadrant. (E, G) OS and DFS were stratified according to all four quadrants defined in D and F and are represented as Kaplan-Meier survival curves with p . CI, confidence interval; DFS, disease-free survival; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; HR, hazard ratio; IHC, immunohistochemistry; LMU, Ludwig-Maximilians-University; OS, overall survival; RPPA, reversed-phase protein atlas; TCGA, the Cancer Genome Atlas.
    Figure Legend Snippet: EGFR and EpCAM expression predicts differential clinical outcome of HNSCCs. (A, C) Expression of EGFR and EpCAM was assessed in serial cryosections of normal mucosa and primary HNSCCs by IHC staining. Shown are representative examples of EGFR high /EpCAM high (A), EGFR high /EpCAM low , and EGFR low /EpCAM high (C) tumors at 100×, 200×, and 400× magnifications. (B) OS probability of HNSCC patients from the LMU cohort ( n = 180) and from a subcohort of the HNSCC TCGA cohort ( n ]. Protein expression data for EGFR from the RPPA data were used for the TCGA cohort. OS is represented as Kaplan-Meier survival curves with p . (D, F) IHC scores of EGFR and EpCAM expression were assessed in n = 180 primary HNSCCs of the LMU cohort (D) and in n = 87 HPV-negative primary HNSCCs of the LMU cohort (F). Expression correlation of EGFR and EpCAM is plotted and subdivided according to a cutoff threshold of 150 (score range 0–300). Numbers and percentages of patients within subgroups are indicated in each quadrant. (E, G) OS and DFS were stratified according to all four quadrants defined in D and F and are represented as Kaplan-Meier survival curves with p . CI, confidence interval; DFS, disease-free survival; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; HR, hazard ratio; IHC, immunohistochemistry; LMU, Ludwig-Maximilians-University; OS, overall survival; RPPA, reversed-phase protein atlas; TCGA, the Cancer Genome Atlas.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    Soluble EpEX-Fc binds to EGFR and induces ERK1/2 and AKT. (A) Bidirectional co-immunoprecipitation (“IP”) of EGFR and EpCAM in whole-cell lysates of FaDu, Cal27, and HCT8 cells using EGFR- and EpCAM-specific antibodies. Isotype control antibody (“IgG”) served as control. Coimmunoprecipitated EGFR and EpCAM were visualized in immunoblotting with specific antibodies (“IB”), with whole-cell lysates as control (“lysate”). Shown are representative results from n = 3 independent experiments. (B) SNs of Cal27, Kyse30, FaDu, and HCT8 cells were immunoprecipitated with EpEX-specific antibodies and separated under reducing (left) and nonreducing native conditions (right), and EpEX was detected with specific antibodies. Antibody HCs and EpEX mono-, di-, and oligomers are indicated. Shown are representative results from n = 3 independent experiments. (C) EpEX-Fc or Fc were incubated with whole-cell lysates of FaDu and Cal27 and immobilized on protein A agarose beads, and protein complexes were separated on SDS-PAGE. Immunoprecipitated proteins were detected by immunoblotting (“IB”) with Fc- and EGFR-specific antibodies. Shown are representative results from n = 3 independent experiments. (D) EGFR ex and EpEX were incubated in the presence or absence of cross-linker (BS3). Where indicated, EGF was added. Monomers, dimers, and EGFR ex /EpEX complexes are marked. Shown are representative results from n = 3 independent experiments. (E, F) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points. Where indicated, cells were additionally treated with Cetuximab (“Cet.”). Phosphorylation of ERK1/2 (E) and AKT (F) was assessed by immunoblotting with specific antibodies. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (G) FaDu and Cal27 cells were kept untreated or were treated with EGF (9 nM), Fc, or EpEX-Fc (10 nM) for 30 min, and phosphorylation of ERK1/2 and AKT was detected by immunofluorescence laser scanning confocal microscopy (ERK1/2 or AKT: green, nuclei: blue [DAPI]). Shown are representative results from n = 3 independent experiments. (H) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points (“EGF 30 min”). Where indicated, MEK1 inhibitor AZD6244 or EGFR inhibitor AG1478 were added. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (I) HEK293 cells were transiently transfected with GFP or EGFR expression plasmids and were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Expression of EGFR and activation of ERK1/2 were assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n ] by immunoblotting. Levels of actin were assessed in parallel. Shown are representative results from n = 3 independent experiments. (K) HCT8WT and CRISPR-Cas9 EpCAM K.O.1 cells were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Activation of ERK1/2 was assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n = 3 independent experiments. BS3, bisulfosuccinimidyl suberate; CRISPR-Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated 9; EGF, epidermal growth factor; EGFR, EGF receptor; EGFR ex , extracellular domain of EGFR; EpCAM, epithelial cell adhesion molecule; EpCAM K.O.1, EPCAM -knockout clone 1; EpEX, extracellular domain of EpCAM; EpICD, intracellular domain of EpCAM; ERK1/2, extracellular signal–regulated kinase 1/2; Fc, fragment crystallizable region; GFP, green fluorescent protein; HC, heavy chain; HCT8WT, HCT8 wild type; HEK293, human embryonic kidney 293; IgG, immunoglobulin G; MW, molecular mass; pAKT, phosphorylated AKT; pERK, phosphorylated ERK; SN, supernatant.
    Figure Legend Snippet: Soluble EpEX-Fc binds to EGFR and induces ERK1/2 and AKT. (A) Bidirectional co-immunoprecipitation (“IP”) of EGFR and EpCAM in whole-cell lysates of FaDu, Cal27, and HCT8 cells using EGFR- and EpCAM-specific antibodies. Isotype control antibody (“IgG”) served as control. Coimmunoprecipitated EGFR and EpCAM were visualized in immunoblotting with specific antibodies (“IB”), with whole-cell lysates as control (“lysate”). Shown are representative results from n = 3 independent experiments. (B) SNs of Cal27, Kyse30, FaDu, and HCT8 cells were immunoprecipitated with EpEX-specific antibodies and separated under reducing (left) and nonreducing native conditions (right), and EpEX was detected with specific antibodies. Antibody HCs and EpEX mono-, di-, and oligomers are indicated. Shown are representative results from n = 3 independent experiments. (C) EpEX-Fc or Fc were incubated with whole-cell lysates of FaDu and Cal27 and immobilized on protein A agarose beads, and protein complexes were separated on SDS-PAGE. Immunoprecipitated proteins were detected by immunoblotting (“IB”) with Fc- and EGFR-specific antibodies. Shown are representative results from n = 3 independent experiments. (D) EGFR ex and EpEX were incubated in the presence or absence of cross-linker (BS3). Where indicated, EGF was added. Monomers, dimers, and EGFR ex /EpEX complexes are marked. Shown are representative results from n = 3 independent experiments. (E, F) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points. Where indicated, cells were additionally treated with Cetuximab (“Cet.”). Phosphorylation of ERK1/2 (E) and AKT (F) was assessed by immunoblotting with specific antibodies. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (G) FaDu and Cal27 cells were kept untreated or were treated with EGF (9 nM), Fc, or EpEX-Fc (10 nM) for 30 min, and phosphorylation of ERK1/2 and AKT was detected by immunofluorescence laser scanning confocal microscopy (ERK1/2 or AKT: green, nuclei: blue [DAPI]). Shown are representative results from n = 3 independent experiments. (H) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points (“EGF 30 min”). Where indicated, MEK1 inhibitor AZD6244 or EGFR inhibitor AG1478 were added. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (I) HEK293 cells were transiently transfected with GFP or EGFR expression plasmids and were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Expression of EGFR and activation of ERK1/2 were assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n ] by immunoblotting. Levels of actin were assessed in parallel. Shown are representative results from n = 3 independent experiments. (K) HCT8WT and CRISPR-Cas9 EpCAM K.O.1 cells were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Activation of ERK1/2 was assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n = 3 independent experiments. BS3, bisulfosuccinimidyl suberate; CRISPR-Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated 9; EGF, epidermal growth factor; EGFR, EGF receptor; EGFR ex , extracellular domain of EGFR; EpCAM, epithelial cell adhesion molecule; EpCAM K.O.1, EPCAM -knockout clone 1; EpEX, extracellular domain of EpCAM; EpICD, intracellular domain of EpCAM; ERK1/2, extracellular signal–regulated kinase 1/2; Fc, fragment crystallizable region; GFP, green fluorescent protein; HC, heavy chain; HCT8WT, HCT8 wild type; HEK293, human embryonic kidney 293; IgG, immunoglobulin G; MW, molecular mass; pAKT, phosphorylated AKT; pERK, phosphorylated ERK; SN, supernatant.

    Techniques Used: Immunoprecipitation, Incubation, SDS Page, Immunofluorescence, Confocal Microscopy, Transfection, Expressing, Activation Assay, CRISPR, Knock-Out

    pERK and Slug are coexpressed and predict poor survival of HNSCC patients. (A) HNSCC tumors were stained for the expression of pERK1/2 and Slug in consecutive cryosections. Shown are 2 examples each of EGFR low /EpCAM high (patients 1 and 2), EGFR high /EpCAM low (patients 3 and 4), and HNSCCs at 100× and 200× magnification. (B) IHC scores of pERK and Slug were compared in EGFR low /EpCAM high ( n = 37) and EGFR high /EpCAM low ( n = 39) HNSCCs. Shown are IHC score values with mean (lines) and Student t . (C) IHC scores of pERK and Slug were compared in a Spearman correlation with r-value and p -value for the entire HNSCC cohort ( n . (D) Patients with EGFR and EpCAM expression levels
    Figure Legend Snippet: pERK and Slug are coexpressed and predict poor survival of HNSCC patients. (A) HNSCC tumors were stained for the expression of pERK1/2 and Slug in consecutive cryosections. Shown are 2 examples each of EGFR low /EpCAM high (patients 1 and 2), EGFR high /EpCAM low (patients 3 and 4), and HNSCCs at 100× and 200× magnification. (B) IHC scores of pERK and Slug were compared in EGFR low /EpCAM high ( n = 37) and EGFR high /EpCAM low ( n = 39) HNSCCs. Shown are IHC score values with mean (lines) and Student t . (C) IHC scores of pERK and Slug were compared in a Spearman correlation with r-value and p -value for the entire HNSCC cohort ( n . (D) Patients with EGFR and EpCAM expression levels

    Techniques Used: Staining, Expressing, Immunohistochemistry

    Schematic representation of EGF and EpEX cross talk at the EGFR. Low-dose EGF induces EGFR activation that results in intermediate ERK1/2 phosphorylation and enhanced cell proliferation (left panel). High-dose EGF induces EGFR activation that results in strong ERK1/2 phosphorylation and induction of EMT, including EMT-TFs Snail, Zeb1, and Slug (center-left panel). High-dose EpEX induces EGFR activation that results in intermediate ERK1/2 phosphorylation and enhanced proliferation (center-right panel); low-dose EpEX has no measurable effect on proliferation (right panel). EGF, epidermal growth factor; EGFR, EGF receptor; EMT, epithelial-mesenchymal transition; EMT-TF, EMT transcription factor; EpEX, extracellular domain of EpCAM; ERK1/2, extracellular signal–regulated kinase; PI3K, phosphoinositide 3-kinase; Zeb1, zinc finger E-box-binding homeobox 1.
    Figure Legend Snippet: Schematic representation of EGF and EpEX cross talk at the EGFR. Low-dose EGF induces EGFR activation that results in intermediate ERK1/2 phosphorylation and enhanced cell proliferation (left panel). High-dose EGF induces EGFR activation that results in strong ERK1/2 phosphorylation and induction of EMT, including EMT-TFs Snail, Zeb1, and Slug (center-left panel). High-dose EpEX induces EGFR activation that results in intermediate ERK1/2 phosphorylation and enhanced proliferation (center-right panel); low-dose EpEX has no measurable effect on proliferation (right panel). EGF, epidermal growth factor; EGFR, EGF receptor; EMT, epithelial-mesenchymal transition; EMT-TF, EMT transcription factor; EpEX, extracellular domain of EpCAM; ERK1/2, extracellular signal–regulated kinase; PI3K, phosphoinositide 3-kinase; Zeb1, zinc finger E-box-binding homeobox 1.

    Techniques Used: Activation Assay, Binding Assay

    EpEX-Fc induces EGFR-dependent proliferation but inhibits high-dose EGF-induced EMT. (A) FaDu and Kyse30 cells were plated at equal numbers and treated with control media, low (1.8 nM) and high (9 nM) doses of EGF, low (1 nM) and high doses (10 nM) of EpEX-Fc, Fc (10 nM), or a combination of EpEX high with Cetuximab (“Cet.”). Cell numbers were assessed after 24, 48, and 72 hr. Shown are means with SDs from n . (B) FaDu and Kyse30 cells were plated at equal numbers and treated with control media, EpEX (10 nM), or a combination of EpEX (10 nM) and Cetuximab (“Cet.”). BrdU incorporation was analyzed after 72 hr. Shown are means with SDs from n . (C) Relative migration of FaDu and Kyse30 cells was assessed in wound healing assays. FaDu, Kyse30, and Cal27 cells were either kept untreated (control) or were treated with Fc (10 nM), EpEX-Fc, EGF, EGF in combination with EpEX (10 nM), or EGF with Cetuximab (“Cet.”). Shown are representative micrograph pictures of cells after 24 hr (Kyse30) and 48 hr (FaDu) from n = 3 independent experiments. (D) Relative migration was quantified from representative micrographs and was normalized for proliferation indexes of each cell line. Shown are means with SDs from n = 3 independent experiments. One-way ANOVA with post hoc multiple testing and Bonferroni correction * p . BrdU, bromodeoxyuridine; EGF, epidermal growth factor; EGFR, EGF receptor; EMT, epithelial-mesenchymal transition; EpEX, extracellular domain of EpCAM; Fc, fragment crystallizable region; ns, not significant; SD, standard deviation.
    Figure Legend Snippet: EpEX-Fc induces EGFR-dependent proliferation but inhibits high-dose EGF-induced EMT. (A) FaDu and Kyse30 cells were plated at equal numbers and treated with control media, low (1.8 nM) and high (9 nM) doses of EGF, low (1 nM) and high doses (10 nM) of EpEX-Fc, Fc (10 nM), or a combination of EpEX high with Cetuximab (“Cet.”). Cell numbers were assessed after 24, 48, and 72 hr. Shown are means with SDs from n . (B) FaDu and Kyse30 cells were plated at equal numbers and treated with control media, EpEX (10 nM), or a combination of EpEX (10 nM) and Cetuximab (“Cet.”). BrdU incorporation was analyzed after 72 hr. Shown are means with SDs from n . (C) Relative migration of FaDu and Kyse30 cells was assessed in wound healing assays. FaDu, Kyse30, and Cal27 cells were either kept untreated (control) or were treated with Fc (10 nM), EpEX-Fc, EGF, EGF in combination with EpEX (10 nM), or EGF with Cetuximab (“Cet.”). Shown are representative micrograph pictures of cells after 24 hr (Kyse30) and 48 hr (FaDu) from n = 3 independent experiments. (D) Relative migration was quantified from representative micrographs and was normalized for proliferation indexes of each cell line. Shown are means with SDs from n = 3 independent experiments. One-way ANOVA with post hoc multiple testing and Bonferroni correction * p . BrdU, bromodeoxyuridine; EGF, epidermal growth factor; EGFR, EGF receptor; EMT, epithelial-mesenchymal transition; EpEX, extracellular domain of EpCAM; Fc, fragment crystallizable region; ns, not significant; SD, standard deviation.

    Techniques Used: BrdU Incorporation Assay, Migration, Standard Deviation

    31) Product Images from "Human cancers converge at the HIF-2? oncogenic axis"

    Article Title: Human cancers converge at the HIF-2? oncogenic axis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0906432106

    HIF-2α activates the EGFR and IGF1R tyrosine kinases and their downstream signaling molecules. ( A ) Relative levels of RTK phosphorylation in serum-starved human cancer cell lines stably expressing scramble control, HIF-1α or HIF-2α
    Figure Legend Snippet: HIF-2α activates the EGFR and IGF1R tyrosine kinases and their downstream signaling molecules. ( A ) Relative levels of RTK phosphorylation in serum-starved human cancer cell lines stably expressing scramble control, HIF-1α or HIF-2α

    Techniques Used: Stable Transfection, Expressing

    32) Product Images from "NF-κB activation is an early event of changes in gene regulation for acquiring drug resistance in human adenocarcinoma PC-9 cells"

    Article Title: NF-κB activation is an early event of changes in gene regulation for acquiring drug resistance in human adenocarcinoma PC-9 cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201796

    Effect of gene silencing against EGFR on NF-κB activation. ( a ) RNAi knockdown. PC-9 cells were subjected to gene silencing with siRNAs against total EGFR (siEGFR) or oncogenic mutant EGFR alleles [si746/50_3D10 (si3D10) targeting the 746/750 deletion in exon 19] in PC-9 cells. Non-silencing siRNAs (siControl: siC) were also examined as a negative control. 24h after introduction of siRNAs, EGFR was examined by western blotting. Alpha-tubulin was examined as an internal control. ( b ) EGFR mRNA levels. Indicated siRNAs were transfected into PC-9 cells as in a . The level of EGFR was examined by RT-qPCR and analyzed by the delta-delta Ct method using the data of Gapdh as a reference. The data were further normalized to the data obtained by siControl (siC) as 1. Data are shown as mean ± SD ( n = 3). ( c ) Activation of NF-κB. The pGL4-NF-κB-RE and phRL-Tk plasmids together with indicated siRNAs (20nM) were introduced into PC-9 cells. After 24h incubation, Dual-luciferase assay was carried out as in Fig 1b ( n = 6). ( d ) Effect of gefitinib on NF-κB activation under EGFR knockdown. Reporter plasmids and siRNAs were introduced into PC9 cells as in c . The cells were treated with 0, 0.1, 1.0 and 10μM of gefitinib (Gef). Dual-luciferase assay was carried out as in Fig 1b ( n = 6). Significant difference in cell viability between gefitinib and DMSO treatment was examined by one-way analysis of variance (ANOVA) (Dunnett’s test; *p
    Figure Legend Snippet: Effect of gene silencing against EGFR on NF-κB activation. ( a ) RNAi knockdown. PC-9 cells were subjected to gene silencing with siRNAs against total EGFR (siEGFR) or oncogenic mutant EGFR alleles [si746/50_3D10 (si3D10) targeting the 746/750 deletion in exon 19] in PC-9 cells. Non-silencing siRNAs (siControl: siC) were also examined as a negative control. 24h after introduction of siRNAs, EGFR was examined by western blotting. Alpha-tubulin was examined as an internal control. ( b ) EGFR mRNA levels. Indicated siRNAs were transfected into PC-9 cells as in a . The level of EGFR was examined by RT-qPCR and analyzed by the delta-delta Ct method using the data of Gapdh as a reference. The data were further normalized to the data obtained by siControl (siC) as 1. Data are shown as mean ± SD ( n = 3). ( c ) Activation of NF-κB. The pGL4-NF-κB-RE and phRL-Tk plasmids together with indicated siRNAs (20nM) were introduced into PC-9 cells. After 24h incubation, Dual-luciferase assay was carried out as in Fig 1b ( n = 6). ( d ) Effect of gefitinib on NF-κB activation under EGFR knockdown. Reporter plasmids and siRNAs were introduced into PC9 cells as in c . The cells were treated with 0, 0.1, 1.0 and 10μM of gefitinib (Gef). Dual-luciferase assay was carried out as in Fig 1b ( n = 6). Significant difference in cell viability between gefitinib and DMSO treatment was examined by one-way analysis of variance (ANOVA) (Dunnett’s test; *p

    Techniques Used: Activation Assay, Mutagenesis, Negative Control, Western Blot, Transfection, Quantitative RT-PCR, Incubation, Luciferase

    33) Product Images from "Pregnancy-upregulated nonubiquitous calmodulin kinase induces ligand-independent EGFR degradation"

    Article Title: Pregnancy-upregulated nonubiquitous calmodulin kinase induces ligand-independent EGFR degradation

    Journal:

    doi: 10.1152/ajpcell.00449.2007

    Upregulation of unliganded EGFR by small interfering (si)RNA-mediated knockdown of endogenous human Pnck in SK-BR-3 cells. A : upregulation of endogenous EGFR by Pnck siRNA. Subconfluent SK-BR-3 breast cancer cells were transfected with either control
    Figure Legend Snippet: Upregulation of unliganded EGFR by small interfering (si)RNA-mediated knockdown of endogenous human Pnck in SK-BR-3 cells. A : upregulation of endogenous EGFR by Pnck siRNA. Subconfluent SK-BR-3 breast cancer cells were transfected with either control

    Techniques Used: Transfection

    Ligand-independent EGFR downregulation by Pnck occurs by protea-lysosomal degradation, independent of EGFR tyrosine kinase activity. A : ligand-independent EGFR downregulation does not occur by transcriptional downregulation of the EGFR gene. Two sets
    Figure Legend Snippet: Ligand-independent EGFR downregulation by Pnck occurs by protea-lysosomal degradation, independent of EGFR tyrosine kinase activity. A : ligand-independent EGFR downregulation does not occur by transcriptional downregulation of the EGFR gene. Two sets

    Techniques Used: Activity Assay

    Ligand-independent EGFR downregulation by Pnck and dissection of MAP kinase signaling in Pnck-expressing HEK-293 cells. A : EGFR undergoes ligand-independent downregulation in HA-Pnck-expressing stable HEK-293 cells. Three dishes each of Neo and HA-Pnck
    Figure Legend Snippet: Ligand-independent EGFR downregulation by Pnck and dissection of MAP kinase signaling in Pnck-expressing HEK-293 cells. A : EGFR undergoes ligand-independent downregulation in HA-Pnck-expressing stable HEK-293 cells. Three dishes each of Neo and HA-Pnck

    Techniques Used: Dissection, Expressing

    Inhibition of total tyrosine, EGFR, and Shc tyrosine phosphorylation by Pnck. A : inhibition of EGF-induced tyrosine phosphorylation in HA-Pnck expressing HEK-293 cells ( lanes 1 – 6 ). Neo ( lanes 1 – 3 ) and HA-Pnck HEK-293 ( lanes 4 –
    Figure Legend Snippet: Inhibition of total tyrosine, EGFR, and Shc tyrosine phosphorylation by Pnck. A : inhibition of EGF-induced tyrosine phosphorylation in HA-Pnck expressing HEK-293 cells ( lanes 1 – 6 ). Neo ( lanes 1 – 3 ) and HA-Pnck HEK-293 ( lanes 4 –

    Techniques Used: Inhibition, Expressing

    34) Product Images from "Oncogenic KRAS-induced epiregulin overexpression contributes to aggressive phenotype and is a promising therapeutic target in non-small-cell lung cancer"

    Article Title: Oncogenic KRAS-induced epiregulin overexpression contributes to aggressive phenotype and is a promising therapeutic target in non-small-cell lung cancer

    Journal: Oncogene

    doi: 10.1038/onc.2012.402

    ( a ) Expression of EREG mRNA in human bronchial epithelial cell lines (noncancerous cells; N = 5), NSCLC cell lines with wild-type EGFR/BRAF/KRAS (EGFR/BRAF/KRAS WT; N = 10), NSCLC cell lines harboring EGFR mutations (EGFR Mut; N = 9), BRAF mutations (BRAF
    Figure Legend Snippet: ( a ) Expression of EREG mRNA in human bronchial epithelial cell lines (noncancerous cells; N = 5), NSCLC cell lines with wild-type EGFR/BRAF/KRAS (EGFR/BRAF/KRAS WT; N = 10), NSCLC cell lines harboring EGFR mutations (EGFR Mut; N = 9), BRAF mutations (BRAF

    Techniques Used: Expressing

    35) Product Images from "High frequency of loss of PTEN expression in human solid salivary adenoid cystic carcinoma and its implication for targeted therapy"

    Article Title: High frequency of loss of PTEN expression in human solid salivary adenoid cystic carcinoma and its implication for targeted therapy

    Journal: Oncotarget

    doi:

    PTEN expression and its correlation with pAKT, pS6, HER2, MYB and EGFR in human salivary gland tumors A. Quantification results of PTEN IHC in human salivary gland tumors. SACC: salivary adenoid cystic carcinomas, PA: pleomorphic adenomas, MEC: mucoepidermoid carcinoma BCC: basal cell carcinoma, MyEC: myoepithelial carcinoma, (BCCs), AIC: acinic cell carcinoma. B. Quantification results of PTEN IHC in three patterns of human SACC. C. Representative images of PTEN, pAKT and pS6 IHC staining in three patterns of human SACCs. The scale bars represent 100 μm. D. Representative images of IHC of HER2, MYB and EGFR in three patterns of human SACCs. The scale bar represents 100 μm. E. Percentage of positive and negative cases of pAKT, pS6, HER2, MYB and EGFR in human SACCs with loss of PTEN expression. Positive of these molecules were defined as the staining index ≥ 3. Loss of PTEN expression was defined as the staining index ≤ 2.
    Figure Legend Snippet: PTEN expression and its correlation with pAKT, pS6, HER2, MYB and EGFR in human salivary gland tumors A. Quantification results of PTEN IHC in human salivary gland tumors. SACC: salivary adenoid cystic carcinomas, PA: pleomorphic adenomas, MEC: mucoepidermoid carcinoma BCC: basal cell carcinoma, MyEC: myoepithelial carcinoma, (BCCs), AIC: acinic cell carcinoma. B. Quantification results of PTEN IHC in three patterns of human SACC. C. Representative images of PTEN, pAKT and pS6 IHC staining in three patterns of human SACCs. The scale bars represent 100 μm. D. Representative images of IHC of HER2, MYB and EGFR in three patterns of human SACCs. The scale bar represents 100 μm. E. Percentage of positive and negative cases of pAKT, pS6, HER2, MYB and EGFR in human SACCs with loss of PTEN expression. Positive of these molecules were defined as the staining index ≥ 3. Loss of PTEN expression was defined as the staining index ≤ 2.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    36) Product Images from "Dissecting the EGFR-PI3K-AKT pathway in oral cancer highlights the role of the EGFR variant III and its clinical relevance"

    Article Title: Dissecting the EGFR-PI3K-AKT pathway in oral cancer highlights the role of the EGFR variant III and its clinical relevance

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-20-43

    Gene copy numbers of the samples. ( A ) A scattergram of the EGFR or PIK3CA results are shown. Each dot represents the specific GCN of the individual specimen analyzed using real-time PCR and the C -2∆tt method. Dots above the upper dotted line have copy number of more than 3. Dots above the lower dashed line have copy number of more than 2. ( B ) An association study of EGFR expression and GCN amplification is shown by the bar chart. The empty and solid bars represent negative or positive GCN amplification. The P level was assessed through Chi-Square analysis.
    Figure Legend Snippet: Gene copy numbers of the samples. ( A ) A scattergram of the EGFR or PIK3CA results are shown. Each dot represents the specific GCN of the individual specimen analyzed using real-time PCR and the C -2∆tt method. Dots above the upper dotted line have copy number of more than 3. Dots above the lower dashed line have copy number of more than 2. ( B ) An association study of EGFR expression and GCN amplification is shown by the bar chart. The empty and solid bars represent negative or positive GCN amplification. The P level was assessed through Chi-Square analysis.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Amplification

    37) Product Images from "Metaplastic breast carcinomas exhibit EGFR, but not HER2, gene amplification and overexpression: immunohistochemical and chromogenic in situ hybridization analysis"

    Article Title: Metaplastic breast carcinomas exhibit EGFR, but not HER2, gene amplification and overexpression: immunohistochemical and chromogenic in situ hybridization analysis

    Journal: Breast Cancer Research

    doi: 10.1186/bcr1341

    EGFR and HER2 overexpression and gene amplification in a spindle cell carcinoma. (a) Photomicrograph of a spindle cell carcinoma (haematoxylin and eosin). Immunohistochemical analysis revealed (b) EGFR grade 3+ positivity and (c) HER2 grade 2+ reactivity. (d) CISH demonstrating EGFR amplification (clusters of signals in the nuclei of neoplastic cells). Note the presence of one or two copies of EGFR in stromal cells (arrowheads). (e) CISH for HER2 gene: no amplification (2–3 gene copies/nucleus). CISH, chromogenic in situ hybridization; EGFR, epidermal growth factor receptor; HER, human epidermal growth factor receptor; MBC, metaplastic breast carcinoma.
    Figure Legend Snippet: EGFR and HER2 overexpression and gene amplification in a spindle cell carcinoma. (a) Photomicrograph of a spindle cell carcinoma (haematoxylin and eosin). Immunohistochemical analysis revealed (b) EGFR grade 3+ positivity and (c) HER2 grade 2+ reactivity. (d) CISH demonstrating EGFR amplification (clusters of signals in the nuclei of neoplastic cells). Note the presence of one or two copies of EGFR in stromal cells (arrowheads). (e) CISH for HER2 gene: no amplification (2–3 gene copies/nucleus). CISH, chromogenic in situ hybridization; EGFR, epidermal growth factor receptor; HER, human epidermal growth factor receptor; MBC, metaplastic breast carcinoma.

    Techniques Used: Over Expression, Amplification, Immunohistochemistry, Chromogenic In Situ Hybridization

    EGFR overexpression and gene amplification in MBCs. Photomicrographs of (a) a spindle cell metaplastic breast carcinoma (haematoxylin and eosin) showing (b) grade 3+ immunohistochemical positivity for EGFR and (c) EGFR gene amplification ( > 5 signals per nucleus [CISH]). Inset in panel c: note the bizarre neoplastic cell with more than 10 copies of EGFR . (d) Breast carcinoma with squamous metaplasia (haematoxylin and eosin) with (e) EGFR grade 3+ immunohistochemical positivity. (f) CISH demonstrating EGFR amplification (clusters of signals in the nuclei of neoplastic cells). Note the presence of one or two signals in the nuclei of stromal cells (arrowheads). CISH, chromogenic in situ hybridization; EGFR, epidermal growth factor receptor; MBC, metaplastic breast carcinoma.
    Figure Legend Snippet: EGFR overexpression and gene amplification in MBCs. Photomicrographs of (a) a spindle cell metaplastic breast carcinoma (haematoxylin and eosin) showing (b) grade 3+ immunohistochemical positivity for EGFR and (c) EGFR gene amplification ( > 5 signals per nucleus [CISH]). Inset in panel c: note the bizarre neoplastic cell with more than 10 copies of EGFR . (d) Breast carcinoma with squamous metaplasia (haematoxylin and eosin) with (e) EGFR grade 3+ immunohistochemical positivity. (f) CISH demonstrating EGFR amplification (clusters of signals in the nuclei of neoplastic cells). Note the presence of one or two signals in the nuclei of stromal cells (arrowheads). CISH, chromogenic in situ hybridization; EGFR, epidermal growth factor receptor; MBC, metaplastic breast carcinoma.

    Techniques Used: Over Expression, Amplification, Immunohistochemistry, Chromogenic In Situ Hybridization

    EGFR overexpression and amplification: prognostic impact on DFS and OS. CISH, chromogenic in situ hybridization; DFS, disease-free survival; EGFR, epidermal growth factor receptor; OS, overall survival.
    Figure Legend Snippet: EGFR overexpression and amplification: prognostic impact on DFS and OS. CISH, chromogenic in situ hybridization; DFS, disease-free survival; EGFR, epidermal growth factor receptor; OS, overall survival.

    Techniques Used: Over Expression, Amplification, Chromogenic In Situ Hybridization

    38) Product Images from "KLF4 defines the efficacy of the epidermal growth factor receptor inhibitor, erlotinib, in triple-negative breast cancer cells by repressing the EGFR gene"

    Article Title: KLF4 defines the efficacy of the epidermal growth factor receptor inhibitor, erlotinib, in triple-negative breast cancer cells by repressing the EGFR gene

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-020-01305-7

    Repression of EGFR is an obligatory intermediate step for KLF4 to inhibit aggressive breast cancer phenotypes. MCF10A cells were transfected with siNS, siKLF4, siEGFR, or siKLF4+siEGFR, and western blots were performed to confirm a KLF4 and b EGFR silencing. Cells were then allowed to c migrate for 6 h or d invade for 16 h before they were stained and counted. e MCF10A cells were transfected with siNS, siKLF4, siEGFR, or siKLF4+siEGFR, and cell number was counted at days 3–5 using trypan blue exclusion assay, * p
    Figure Legend Snippet: Repression of EGFR is an obligatory intermediate step for KLF4 to inhibit aggressive breast cancer phenotypes. MCF10A cells were transfected with siNS, siKLF4, siEGFR, or siKLF4+siEGFR, and western blots were performed to confirm a KLF4 and b EGFR silencing. Cells were then allowed to c migrate for 6 h or d invade for 16 h before they were stained and counted. e MCF10A cells were transfected with siNS, siKLF4, siEGFR, or siKLF4+siEGFR, and cell number was counted at days 3–5 using trypan blue exclusion assay, * p

    Techniques Used: Transfection, Western Blot, Staining, Trypan Blue Exclusion Assay

    KLF4 expression dictates sensitivity to pharmacological inhibition of EGFR. a MDA-MB-231 and b MDA-MB-468 cells were infected with AdGFP or AdKLF4, and 2 days later, treated with 0–60 μM erlotinib for 3 days. The live cells were counted using trypan blue exclusion assay. Responses were normalized to effects observed with no drug for both AdGFP- and AdKLF4-infected cells. c MCF10A cells were transfected with siNS or siKLF4, and after 1 day, were treated with 0–60 μM erlotinib for 3 days. Cell number was counted, and the relative impact of the drug was normalized for siNS or siKLF4 in the absence of the drug. For each graph, nonlinear regression analysis was performed on IC 50 values of control versus experimental group, and each comparison resulted in statistical significance at p
    Figure Legend Snippet: KLF4 expression dictates sensitivity to pharmacological inhibition of EGFR. a MDA-MB-231 and b MDA-MB-468 cells were infected with AdGFP or AdKLF4, and 2 days later, treated with 0–60 μM erlotinib for 3 days. The live cells were counted using trypan blue exclusion assay. Responses were normalized to effects observed with no drug for both AdGFP- and AdKLF4-infected cells. c MCF10A cells were transfected with siNS or siKLF4, and after 1 day, were treated with 0–60 μM erlotinib for 3 days. Cell number was counted, and the relative impact of the drug was normalized for siNS or siKLF4 in the absence of the drug. For each graph, nonlinear regression analysis was performed on IC 50 values of control versus experimental group, and each comparison resulted in statistical significance at p

    Techniques Used: Expressing, Inhibition, Multiple Displacement Amplification, Infection, Trypan Blue Exclusion Assay, Transfection

    Identification of a KLF4-regulated protein signature reveals EGFR as a downstream target. a Western blot confirming the upregulation of KLF4 and its downstream target, E-cadherin, in MDA-MB-231 cells after AdGFP or AdKLF4 infection. b Reverse phase protein array heatmap showing differentially expressed genes after AdGFP or AdKLF4 infection in MDA-MB-231 cells. This analysis was completed as one experiment in duplicate. Proteins whose expression fold change was significantly different ( p
    Figure Legend Snippet: Identification of a KLF4-regulated protein signature reveals EGFR as a downstream target. a Western blot confirming the upregulation of KLF4 and its downstream target, E-cadherin, in MDA-MB-231 cells after AdGFP or AdKLF4 infection. b Reverse phase protein array heatmap showing differentially expressed genes after AdGFP or AdKLF4 infection in MDA-MB-231 cells. This analysis was completed as one experiment in duplicate. Proteins whose expression fold change was significantly different ( p

    Techniques Used: Western Blot, Multiple Displacement Amplification, Infection, Protein Array, Expressing

    KLF4 represses transcription of the EGFR gene. a RT-qPCR quantitation of EGFR mRNA levels following infection of MDA-MB-231 (231) and MDA-MB-468 (468) cells with AdGFP or AdKLF4. b RT-qPCR analysis of EGFR mRNA after silencing KLF4 in MCF10A cells. c Schematic of EGFR promoter-specific sites (1–6) used to detect KLF4 binding by ChIP-PCR. Three primer sets (A/B, C/D, and E/F) were used as indicated in the schematic and labels. Primers are listed in Table S 1 . d Gene-specific ChIP-PCR gel of MCF10A cells assessing the binding of KLF4 protein to the EGFR gene locus (hg19) where K1 and K2 are technical replicates for KLF4 immunoprecipitation. e Quantitation of d relative to the input. a , b Completed in three independent experiments in triplicate with * p
    Figure Legend Snippet: KLF4 represses transcription of the EGFR gene. a RT-qPCR quantitation of EGFR mRNA levels following infection of MDA-MB-231 (231) and MDA-MB-468 (468) cells with AdGFP or AdKLF4. b RT-qPCR analysis of EGFR mRNA after silencing KLF4 in MCF10A cells. c Schematic of EGFR promoter-specific sites (1–6) used to detect KLF4 binding by ChIP-PCR. Three primer sets (A/B, C/D, and E/F) were used as indicated in the schematic and labels. Primers are listed in Table S 1 . d Gene-specific ChIP-PCR gel of MCF10A cells assessing the binding of KLF4 protein to the EGFR gene locus (hg19) where K1 and K2 are technical replicates for KLF4 immunoprecipitation. e Quantitation of d relative to the input. a , b Completed in three independent experiments in triplicate with * p

    Techniques Used: Quantitative RT-PCR, Quantitation Assay, Infection, Multiple Displacement Amplification, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation

    KLF4 represses the EGFR signaling pathway. a Western blot analysis of KLF4, tEGFR, pEGFR (Y1068), tAKT, pAKT (S473), tERK1/2, and pERK1/2 (Y202/Y204) protein levels 3 days after AdGFP or AdKLF4 infection of MDA-MB-231 cells. b Graph depicting western blot quantification. All protein levels were normalized to total protein in the lane to control for loading, and phosphorylated proteins were normalized to their respective unphosphorylated proteins. AdKLF4 lanes were then expressed relative to AdGFP and are graphed with the horizontal bars indicating the median of the values of three replicate blots. Each dot represents the mean value from a different experiment, * p
    Figure Legend Snippet: KLF4 represses the EGFR signaling pathway. a Western blot analysis of KLF4, tEGFR, pEGFR (Y1068), tAKT, pAKT (S473), tERK1/2, and pERK1/2 (Y202/Y204) protein levels 3 days after AdGFP or AdKLF4 infection of MDA-MB-231 cells. b Graph depicting western blot quantification. All protein levels were normalized to total protein in the lane to control for loading, and phosphorylated proteins were normalized to their respective unphosphorylated proteins. AdKLF4 lanes were then expressed relative to AdGFP and are graphed with the horizontal bars indicating the median of the values of three replicate blots. Each dot represents the mean value from a different experiment, * p

    Techniques Used: Western Blot, Infection, Multiple Displacement Amplification

    39) Product Images from "HER2 and EGFR overexpression support metastatic progression of prostate cancer to bone"

    Article Title: HER2 and EGFR overexpression support metastatic progression of prostate cancer to bone

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-16-1656

    Analysis of circulating tumor cells (CTCs) from 10 patients with metastatic prostate cancer A , Cluster of white blood cells surrounding a CK+/EGFR+ CTC from patient 3. White blood cells stained positive for CD45. B,C , Other representative images of CK+/EGFR+ CTCs from patients 3 and 5 respectively. D , Clinical history of prostate cancer patients analyzed for circulating tumor cells. E , Number of cytokeratin (CK)+/EGFR+ and CK+/EGFR− CTCs isolated per 1mL whole blood. Numbers above the columns indicate the total number of CTCs isolated from patients 1–10.
    Figure Legend Snippet: Analysis of circulating tumor cells (CTCs) from 10 patients with metastatic prostate cancer A , Cluster of white blood cells surrounding a CK+/EGFR+ CTC from patient 3. White blood cells stained positive for CD45. B,C , Other representative images of CK+/EGFR+ CTCs from patients 3 and 5 respectively. D , Clinical history of prostate cancer patients analyzed for circulating tumor cells. E , Number of cytokeratin (CK)+/EGFR+ and CK+/EGFR− CTCs isolated per 1mL whole blood. Numbers above the columns indicate the total number of CTCs isolated from patients 1–10.

    Techniques Used: Staining, Isolation

    40) Product Images from "Genetic and Expression Analysis of HER-2 and EGFR Genes in Salivary Duct Carcinoma: Empirical and Therapeutic Significance"

    Article Title: Genetic and Expression Analysis of HER-2 and EGFR Genes in Salivary Duct Carcinoma: Empirical and Therapeutic Significance

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-09-0238

    Two salivary duct carcinomas with A, B) Chromosome 7 polysomy and C,D) HER-2 amplification. (A, C) Hematoxylin and eosin sections (10x) of ductal growth pattern; (B) Strong EGFR membranous expression by immunohistochemistry (IHC); (B inset) FISH evaluation:
    Figure Legend Snippet: Two salivary duct carcinomas with A, B) Chromosome 7 polysomy and C,D) HER-2 amplification. (A, C) Hematoxylin and eosin sections (10x) of ductal growth pattern; (B) Strong EGFR membranous expression by immunohistochemistry (IHC); (B inset) FISH evaluation:

    Techniques Used: Amplification, Expressing, Immunohistochemistry, Fluorescence In Situ Hybridization

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Pharmacologically Controlled Protein Switch for ON-OFF Regulation of Growth Factor Activity
    Article Snippet: .. VEGFR2 (Life Technologies, Karlsruhe, Germany, cat. no. PV3660) was diluted in Tris-buffered saline (TBS; 50 mM Tris pH 7.8 and 150 mM NaCl) to a final concentration of 4 ng ml−1 and 100 μl were added to each well of a 96-well ELISA plate (Corning, Lowell, MA, cat. no. 3590) followed by overnight incubation at room temperature. ..

    Transfection:

    Article Title: Editing VEGFR2 Blocks VEGF-Induced Activation of Akt and Tube Formation
    Article Snippet: .. Cell Culture and Transfection Porcine aortic endothelial (PAE) cells overexpressing human VEGFR2 (PAE-KDR cells) , were cultured in a 1:1 mixture of DMEM and Ham's F-12 Nutrient Mixture (Thermo Fisher Scientific, Grand Island, NY, USA), which was supplemented with 10% fetal bovine serum (FBS; Lonza, Walkersville, MD, USA), 100 U/mL penicillin G, and 100 mg/mL streptomycin (Gemini BioProducts, West Sacramento, CA, USA). .. Primary human retinal microvascular endothelial cells (HRECs) were purchased from Cell Systems (Kirkland, WA, USA) and cultured in an endothelial growth medium (EGM)-2 kit (Lonza).

    shRNA:

    Article Title: An apical actin-rich domain drives the establishment of cell polarity during cell adhesion
    Article Snippet: .. RNA interference-mediated knock down of VEGFR-2 and Moesin Silencing experiments were performed using pLKO.1 retroviral vectors from TRC lentiviral shRNA libraries expressing specific shRNAs for human VEGFR-2 (Open Biosystems; clone ID: TRCN0000001685, referred to as 85, and TRCN0000001686, referred to as 86), and Moesin (Sigma-Aldrich; clone ID: TCRN0000344732, validated by the company and referred to as M32). .. Recombinant lentiviruses were produced and used for infection experiments as previously described (Orlandini et al. ).

    Cell Culture:

    Article Title: Editing VEGFR2 Blocks VEGF-Induced Activation of Akt and Tube Formation
    Article Snippet: .. Cell Culture and Transfection Porcine aortic endothelial (PAE) cells overexpressing human VEGFR2 (PAE-KDR cells) , were cultured in a 1:1 mixture of DMEM and Ham's F-12 Nutrient Mixture (Thermo Fisher Scientific, Grand Island, NY, USA), which was supplemented with 10% fetal bovine serum (FBS; Lonza, Walkersville, MD, USA), 100 U/mL penicillin G, and 100 mg/mL streptomycin (Gemini BioProducts, West Sacramento, CA, USA). .. Primary human retinal microvascular endothelial cells (HRECs) were purchased from Cell Systems (Kirkland, WA, USA) and cultured in an endothelial growth medium (EGM)-2 kit (Lonza).

    Real-time Polymerase Chain Reaction:

    Article Title: Catecholamines facilitate VEGF-dependent angiogenesis via β2-adrenoceptor-induced Epac1 and PKA activation
    Article Snippet: .. Real-time PCR was performed in triplicates by using master mix (KK4705, Kapa Biosystems) and the Taqman probes for either VEGF (Hs00900055, Invitrogen) or VEGFR-2 (Hs00911700, Invitrogen), while RPL10 (Hs00749196, Invitrogen) was used as an internal control. .. A program of 50 °C x 2 min, 95 °C x 10 min followed by 40 cycles of 95 °C x 15 sec and 60 °C x 1 min was performed on Applied Biosystem's Step-One Plus Real-time PCR thermocycler in a 96-well plate format.

    Concentration Assay:

    Article Title: Pharmacologically Controlled Protein Switch for ON-OFF Regulation of Growth Factor Activity
    Article Snippet: .. VEGFR2 (Life Technologies, Karlsruhe, Germany, cat. no. PV3660) was diluted in Tris-buffered saline (TBS; 50 mM Tris pH 7.8 and 150 mM NaCl) to a final concentration of 4 ng ml−1 and 100 μl were added to each well of a 96-well ELISA plate (Corning, Lowell, MA, cat. no. 3590) followed by overnight incubation at room temperature. ..

    Incubation:

    Article Title: Pharmacologically Controlled Protein Switch for ON-OFF Regulation of Growth Factor Activity
    Article Snippet: .. VEGFR2 (Life Technologies, Karlsruhe, Germany, cat. no. PV3660) was diluted in Tris-buffered saline (TBS; 50 mM Tris pH 7.8 and 150 mM NaCl) to a final concentration of 4 ng ml−1 and 100 μl were added to each well of a 96-well ELISA plate (Corning, Lowell, MA, cat. no. 3590) followed by overnight incubation at room temperature. ..

    Expressing:

    Article Title: ENDOTHELIAL PROGENITOR CELL NUMBER AND COLONY-FORMING CAPACITY IN OVERWEIGHT AND OBESE ADULTS
    Article Snippet: .. Endothelial phenotype of these cells was confirmed by immunofluorescent staining for the uptake of DiI-ac-LDL (Biomedical Technologies Inc., Stoughton, MA, USA) and expression of von Willebrand factor (Dako, Glostrup, Denmark), VE-cadherin, CD31, and VEGFR-2 (Invitrogen, Carlsbad, CA, USA). .. EPC Number Circulating putative EPC number was determined by fluorescence-activated cell sorting (FACS) analysis following guidelines recommended by the International Society for Hematotherapy and Graft Engineering ( ).

    Article Title: Aging Is Associated with a Proapoptotic Endothelial Progenitor Cell Phenotype
    Article Snippet: .. Endothelial phenotype of these putative EPCs was confirmed by immunofluorescent staining for the uptake of DiI-ac-LDL (Biomedical Technologies, Stoughton, Mass., USA) and expression of von Willebrand factor (Dako, Glostrup, Denmark), VE-cadherin, CD31 and VEGFR-2 (Invitrogen, Carlsbad, Calif., USA). .. In addition, fluorescence-activated cell-sorting analysis utilizing antibodies recognizing cell surface expression of VEGFR-2 (R & D Systems, Minneapolis, Minn., USA), CD34 (Beckman Coulter, Fullerton, Calif., USA) and CD133 (Miltenyi Biotech, Auburn, Calif., USA) were performed in selected samples.

    Article Title: An apical actin-rich domain drives the establishment of cell polarity during cell adhesion
    Article Snippet: .. RNA interference-mediated knock down of VEGFR-2 and Moesin Silencing experiments were performed using pLKO.1 retroviral vectors from TRC lentiviral shRNA libraries expressing specific shRNAs for human VEGFR-2 (Open Biosystems; clone ID: TRCN0000001685, referred to as 85, and TRCN0000001686, referred to as 86), and Moesin (Sigma-Aldrich; clone ID: TCRN0000344732, validated by the company and referred to as M32). .. Recombinant lentiviruses were produced and used for infection experiments as previously described (Orlandini et al. ).

    Staining:

    Article Title: ENDOTHELIAL PROGENITOR CELL NUMBER AND COLONY-FORMING CAPACITY IN OVERWEIGHT AND OBESE ADULTS
    Article Snippet: .. Endothelial phenotype of these cells was confirmed by immunofluorescent staining for the uptake of DiI-ac-LDL (Biomedical Technologies Inc., Stoughton, MA, USA) and expression of von Willebrand factor (Dako, Glostrup, Denmark), VE-cadherin, CD31, and VEGFR-2 (Invitrogen, Carlsbad, CA, USA). .. EPC Number Circulating putative EPC number was determined by fluorescence-activated cell sorting (FACS) analysis following guidelines recommended by the International Society for Hematotherapy and Graft Engineering ( ).

    Article Title: Aging Is Associated with a Proapoptotic Endothelial Progenitor Cell Phenotype
    Article Snippet: .. Endothelial phenotype of these putative EPCs was confirmed by immunofluorescent staining for the uptake of DiI-ac-LDL (Biomedical Technologies, Stoughton, Mass., USA) and expression of von Willebrand factor (Dako, Glostrup, Denmark), VE-cadherin, CD31 and VEGFR-2 (Invitrogen, Carlsbad, Calif., USA). .. In addition, fluorescence-activated cell-sorting analysis utilizing antibodies recognizing cell surface expression of VEGFR-2 (R & D Systems, Minneapolis, Minn., USA), CD34 (Beckman Coulter, Fullerton, Calif., USA) and CD133 (Miltenyi Biotech, Auburn, Calif., USA) were performed in selected samples.

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    Thermo Fisher anti egfr antibody
    Baculovirus-expressing <t>EGFR</t> production. A , Map of the EGFR baculovirus using the flashBACULTRA system with a FLAG epitope placed between the leader sequence peptide (Lrig1 SS) and the full-length mammalian EGFR sequence (EGFR). B , Sf9 insect cells were infected with a MOI of 10 of control baculovirus (C) or EGFR baculovirus (E) for 48 hours followed by collection of total cell lysate and Western Blotting to detect EGFR expression. A set of these cells was treated with 10 ng/ml EGF in order to detect ligand-induced receptor phosphorylation. C , Sf9 insect cells were seeded on glass coverslips and infected with an MOI of 10 of control (C) or EGFR (E) baculovirus for 48 hours. Cells were fixed in 4% paraformaldehyde followed by immunostaining with anti-EGFR antibody (in red) with (+P) or without (-P) permeabilization in 100% methanol. The brightfield image shows cell morphology. Cells incubated with secondary antibody in the absence of EGFR antibody (no primary) were used as a negative control. D , FLAG-EGFR is readily purified from Sf9 insect cells as shown by a representative image of anti-EGFR immunoblot of FLAG EGFR purification and E , a Coomassie-stained SDS-PAGE gel. Bovine serum albumin (BSA) was loaded as a protein of known concentration. Total insect cell lysate starting material (SM), purification washes 1, 3 (W1, W3), and elution (E) were loaded to show efficiency of the FLAG-purification.
    Anti Egfr Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher acp tagged egfr expressing cho cells
    NSCLC-associated <t>EGFR</t> kinase domain mutants are constitutively active and show increased phosphorylation with increasing receptor expression. (A) <t>CHO</t> cells expressing HA-tagged EGFR (HA-EGFR): EGFR-WT, EGFR-L858R, or EGFR-ΔL747-P753insS were serum starved; this was followed by treatment without and with EGF. Cells were pretreated with TKI PD153035 (1 μM) as indicated. Lysates were probed for phosphorylated EGFR (top) as well as total EGFR (bottom). (B) CHO cells expressing the indicated EGFR construct were labeled with an FITC-labeled α-HA Fab (green), then fixed, permeabilized, and labeled using α-pY1068 (red). (C) Cells imaged as in B were quantified for EGFR expression and phosphorylation. Each data point represents the mean pY1068 fluorescence intensity for a given EGFR intensity value per pixel across multiple images after thresholding (arrowhead and dashed line) to eliminate contribution from cells with no detectable EGFR expression; error bars illustrate the SD of the measurement. Solid curves were obtained from model 1 (Eqs. 5 and 6 with the constraints on parameter values indicated in the Supplemental Methods); these curves reflect differences in the kinase activities of mutant and WT forms of EGFR. Dashed curves were obtained from model 2 (Eqs. 5 and 6 with the constraints on parameter values indicated in the Supplemental Methods); these curves reflect differences in dimerization affinities of mutant and WT forms of EGFR. Best-fit values for models 1 and 2 and confidence limits from bootstrapping are given in Supplemental Table S3. Individual plots for EGFR-WT, EGFR-L858R, and EGFR- ΔL747-P753insS are shown in Supplemental Figure S2. A complete description each model is provided in the Supplementary Note.
    Acp Tagged Egfr Expressing Cho Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher egfr mutations
    <t>EGFR</t> mutations detected by three platforms using tumor tissue and plasma samples A heatmap representation of mutations detected from paired tumor tissue/pleural effusion and plasma samples, which were collected from 47 patients. The sample types and platforms used are shown in the left column. Forty-seven tumor tissue samples were detected using ARMS <t>PCR,</t> while 42 tumor samples and 6 pleural effusion samples were detected using Proton and the digital PCR platform. Patients with tumor tissue samples in ARMS PCR, but with pleural effusion samples in Proton and digital PCR were marked with the symbol “PE”. P42 had both tumor tissue and pleural effusion samples detected in Proton and digital PCR. Forty-seven paired plasma samples were detected using digital PCR only. The top two rows show patients’ gender and clinical stage, the upper middle three rows show tumor tissue samples tested by ARMS PCR, the middle six rows show tumor tissue or pleural effusion samples tested by Proton and digital PCR, and the bottom three rows show plasma samples tested by digital PCR only. Mutation events are represented by different colors, e.g., L858R by green, T790M by yellow, 19del by brown, and wild-type mutations by gray. The sites of ctDNA that were not detected are shown as blank.
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    Baculovirus-expressing EGFR production. A , Map of the EGFR baculovirus using the flashBACULTRA system with a FLAG epitope placed between the leader sequence peptide (Lrig1 SS) and the full-length mammalian EGFR sequence (EGFR). B , Sf9 insect cells were infected with a MOI of 10 of control baculovirus (C) or EGFR baculovirus (E) for 48 hours followed by collection of total cell lysate and Western Blotting to detect EGFR expression. A set of these cells was treated with 10 ng/ml EGF in order to detect ligand-induced receptor phosphorylation. C , Sf9 insect cells were seeded on glass coverslips and infected with an MOI of 10 of control (C) or EGFR (E) baculovirus for 48 hours. Cells were fixed in 4% paraformaldehyde followed by immunostaining with anti-EGFR antibody (in red) with (+P) or without (-P) permeabilization in 100% methanol. The brightfield image shows cell morphology. Cells incubated with secondary antibody in the absence of EGFR antibody (no primary) were used as a negative control. D , FLAG-EGFR is readily purified from Sf9 insect cells as shown by a representative image of anti-EGFR immunoblot of FLAG EGFR purification and E , a Coomassie-stained SDS-PAGE gel. Bovine serum albumin (BSA) was loaded as a protein of known concentration. Total insect cell lysate starting material (SM), purification washes 1, 3 (W1, W3), and elution (E) were loaded to show efficiency of the FLAG-purification.

    Journal: PLoS ONE

    Article Title: Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system

    doi: 10.1371/journal.pone.0177761

    Figure Lengend Snippet: Baculovirus-expressing EGFR production. A , Map of the EGFR baculovirus using the flashBACULTRA system with a FLAG epitope placed between the leader sequence peptide (Lrig1 SS) and the full-length mammalian EGFR sequence (EGFR). B , Sf9 insect cells were infected with a MOI of 10 of control baculovirus (C) or EGFR baculovirus (E) for 48 hours followed by collection of total cell lysate and Western Blotting to detect EGFR expression. A set of these cells was treated with 10 ng/ml EGF in order to detect ligand-induced receptor phosphorylation. C , Sf9 insect cells were seeded on glass coverslips and infected with an MOI of 10 of control (C) or EGFR (E) baculovirus for 48 hours. Cells were fixed in 4% paraformaldehyde followed by immunostaining with anti-EGFR antibody (in red) with (+P) or without (-P) permeabilization in 100% methanol. The brightfield image shows cell morphology. Cells incubated with secondary antibody in the absence of EGFR antibody (no primary) were used as a negative control. D , FLAG-EGFR is readily purified from Sf9 insect cells as shown by a representative image of anti-EGFR immunoblot of FLAG EGFR purification and E , a Coomassie-stained SDS-PAGE gel. Bovine serum albumin (BSA) was loaded as a protein of known concentration. Total insect cell lysate starting material (SM), purification washes 1, 3 (W1, W3), and elution (E) were loaded to show efficiency of the FLAG-purification.

    Article Snippet: Cells were washed in PBS, blocked in 5% BSA, incubated with anti-EGFR antibody followed by anti-rabbit Alexa Flour 555 antibody (Thermo Fisher), mounted with fluoromount G (SouthernBiotech), and imaged.

    Techniques: Expressing, FLAG-tag, Sequencing, Infection, Western Blot, Immunostaining, Incubation, Negative Control, Purification, Staining, SDS Page, Concentration Assay

    EGFR incorporation in self-assembled NLPs. A , P1 and P2 SEC fractions were collected and spotted onto nitrocellulose membrane followed by immunoblotting with anti-ApoA1 and anti-EGFR antibodies showing that the majority of EGFR is incorporated into the higher molecular weight NLPs of the P1 fraction. B , Indicated NLPs were separated by denaturing SDS-PAGE and analyzed with anti-pEGFR, anti-EGFR, and anti-ApoA1 antibodies. Numbers indicate band intensity relative to EGFR-NLP normalized to ApoA1 signal. C , Purified Empty and EGFR-NLPs were separated by NativePAGE gels and immunostained with anti-pY20 and anti-EGFR antibodies showing EGFR expression in the higher molecular weight ranges above 480 kDa. D , Bar graph of determination of EGFR insertion into NLP. Total amount of EGFR in the NLP assembly mixture and purified EGFR-NLPs were determined by ELISA and EGFR insertion rate indicated. The values represent the mean ± standard error of the mean (SEM) for two technical replicates. E , EGFR-NLPs were extracted with PBS (control) or sodium carbonate (Na 2 CO 3 ), centrifuged to separate the supernatant containing NLPs (S) and pellet containing insoluble free protein (P), separated by denaturing SDS-PAGE, and analyzed with anti-EGFR antibody showing that EGFR is not susceptible to carbonate extraction.

    Journal: PLoS ONE

    Article Title: Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system

    doi: 10.1371/journal.pone.0177761

    Figure Lengend Snippet: EGFR incorporation in self-assembled NLPs. A , P1 and P2 SEC fractions were collected and spotted onto nitrocellulose membrane followed by immunoblotting with anti-ApoA1 and anti-EGFR antibodies showing that the majority of EGFR is incorporated into the higher molecular weight NLPs of the P1 fraction. B , Indicated NLPs were separated by denaturing SDS-PAGE and analyzed with anti-pEGFR, anti-EGFR, and anti-ApoA1 antibodies. Numbers indicate band intensity relative to EGFR-NLP normalized to ApoA1 signal. C , Purified Empty and EGFR-NLPs were separated by NativePAGE gels and immunostained with anti-pY20 and anti-EGFR antibodies showing EGFR expression in the higher molecular weight ranges above 480 kDa. D , Bar graph of determination of EGFR insertion into NLP. Total amount of EGFR in the NLP assembly mixture and purified EGFR-NLPs were determined by ELISA and EGFR insertion rate indicated. The values represent the mean ± standard error of the mean (SEM) for two technical replicates. E , EGFR-NLPs were extracted with PBS (control) or sodium carbonate (Na 2 CO 3 ), centrifuged to separate the supernatant containing NLPs (S) and pellet containing insoluble free protein (P), separated by denaturing SDS-PAGE, and analyzed with anti-EGFR antibody showing that EGFR is not susceptible to carbonate extraction.

    Article Snippet: Cells were washed in PBS, blocked in 5% BSA, incubated with anti-EGFR antibody followed by anti-rabbit Alexa Flour 555 antibody (Thermo Fisher), mounted with fluoromount G (SouthernBiotech), and imaged.

    Techniques: Size-exclusion Chromatography, Molecular Weight, SDS Page, Purification, Expressing, Enzyme-linked Immunosorbent Assay

    Representative immunofluorescence images of ReNcells and GBMs, in standalone and cocultures, on day 10. For comparison, in select cases, ReNcells cultures in the presence of bFGF were also stained and imaged after 24 h in culture. Cultures were counterstained with DAPI for cell identification. Primary antibodies for TUJ1, GFAP, Nestin, CD133, EGFR, MAP2, and β-actin were used, with appropriate secondary antibodies. Double-immunolabeling was performed under select conditions. Scale bar: 50 μm.

    Journal: Experimental cell research

    Article Title: Pediatric glioblastoma cells inhibit neurogenesis and promote astrogenesis, phenotypic transformation and migration of human neural progenitor cells within cocultures

    doi: 10.1016/j.yexcr.2017.11.013

    Figure Lengend Snippet: Representative immunofluorescence images of ReNcells and GBMs, in standalone and cocultures, on day 10. For comparison, in select cases, ReNcells cultures in the presence of bFGF were also stained and imaged after 24 h in culture. Cultures were counterstained with DAPI for cell identification. Primary antibodies for TUJ1, GFAP, Nestin, CD133, EGFR, MAP2, and β-actin were used, with appropriate secondary antibodies. Double-immunolabeling was performed under select conditions. Scale bar: 50 μm.

    Article Snippet: After removing the blocking buffer, cells were incubated with respective primary antibodies (4 °C, 24 h): rabbit monoclonal anti-TUJ1 (Abcam), mouse monoclonal anti-Nestin (Millipore), mouse monoclonal anti-CD133 (Prominin-1; glioblastoma and stem cell glycoprotein; Millipore), goat polyclonal anti-GFAP (Abcam), mouse monoclonal anti-SOX2 (ThermoFisher Scientific), rabbit monoclonal anti-EGFR (ThermoFisher Scientific), chicken monoclonal anti-MAP2 (ThermoFisher Scientific) and actin-staining Alexa Flour™ 488 Phalloidin (ThermoFisher Scientific).

    Techniques: Immunofluorescence, Staining, Immunolabeling

    NSCLC-associated EGFR kinase domain mutants are constitutively active and show increased phosphorylation with increasing receptor expression. (A) CHO cells expressing HA-tagged EGFR (HA-EGFR): EGFR-WT, EGFR-L858R, or EGFR-ΔL747-P753insS were serum starved; this was followed by treatment without and with EGF. Cells were pretreated with TKI PD153035 (1 μM) as indicated. Lysates were probed for phosphorylated EGFR (top) as well as total EGFR (bottom). (B) CHO cells expressing the indicated EGFR construct were labeled with an FITC-labeled α-HA Fab (green), then fixed, permeabilized, and labeled using α-pY1068 (red). (C) Cells imaged as in B were quantified for EGFR expression and phosphorylation. Each data point represents the mean pY1068 fluorescence intensity for a given EGFR intensity value per pixel across multiple images after thresholding (arrowhead and dashed line) to eliminate contribution from cells with no detectable EGFR expression; error bars illustrate the SD of the measurement. Solid curves were obtained from model 1 (Eqs. 5 and 6 with the constraints on parameter values indicated in the Supplemental Methods); these curves reflect differences in the kinase activities of mutant and WT forms of EGFR. Dashed curves were obtained from model 2 (Eqs. 5 and 6 with the constraints on parameter values indicated in the Supplemental Methods); these curves reflect differences in dimerization affinities of mutant and WT forms of EGFR. Best-fit values for models 1 and 2 and confidence limits from bootstrapping are given in Supplemental Table S3. Individual plots for EGFR-WT, EGFR-L858R, and EGFR- ΔL747-P753insS are shown in Supplemental Figure S2. A complete description each model is provided in the Supplementary Note.

    Journal: Molecular Biology of the Cell

    Article Title: Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer

    doi: 10.1091/mbc.E15-05-0269

    Figure Lengend Snippet: NSCLC-associated EGFR kinase domain mutants are constitutively active and show increased phosphorylation with increasing receptor expression. (A) CHO cells expressing HA-tagged EGFR (HA-EGFR): EGFR-WT, EGFR-L858R, or EGFR-ΔL747-P753insS were serum starved; this was followed by treatment without and with EGF. Cells were pretreated with TKI PD153035 (1 μM) as indicated. Lysates were probed for phosphorylated EGFR (top) as well as total EGFR (bottom). (B) CHO cells expressing the indicated EGFR construct were labeled with an FITC-labeled α-HA Fab (green), then fixed, permeabilized, and labeled using α-pY1068 (red). (C) Cells imaged as in B were quantified for EGFR expression and phosphorylation. Each data point represents the mean pY1068 fluorescence intensity for a given EGFR intensity value per pixel across multiple images after thresholding (arrowhead and dashed line) to eliminate contribution from cells with no detectable EGFR expression; error bars illustrate the SD of the measurement. Solid curves were obtained from model 1 (Eqs. 5 and 6 with the constraints on parameter values indicated in the Supplemental Methods); these curves reflect differences in the kinase activities of mutant and WT forms of EGFR. Dashed curves were obtained from model 2 (Eqs. 5 and 6 with the constraints on parameter values indicated in the Supplemental Methods); these curves reflect differences in dimerization affinities of mutant and WT forms of EGFR. Best-fit values for models 1 and 2 and confidence limits from bootstrapping are given in Supplemental Table S3. Individual plots for EGFR-WT, EGFR-L858R, and EGFR- ΔL747-P753insS are shown in Supplemental Figure S2. A complete description each model is provided in the Supplementary Note.

    Article Snippet: The ACP-tagged EGFR-expressing CHO cells were plated and grown in four-well Lab-Tek chambered coverglass slides (Thermo Fisher) in DMEM with 10% fetal calf serum.

    Techniques: Expressing, Construct, Labeling, Fluorescence, Mutagenesis

    FRET-FLIM reveals that unliganded EGFR-L858R adopts the extended conformation and requires ectodomain interactions for signaling. (A) Schematic of the EGFR ectodomain in the tethered, autoinhibited conformation (left) and the extended conformation (right). The donor fluorophore (Oregon Green 488, green) is covalently linked at the EGFR N-terminus via a small ACP tag, and the acceptor fluorophore (NR12S, a derivate of Nile Red, red) is embedded in the outer leaflet of the plasma membrane. The relative apparent separation between the EGFR N-terminus and the plasma membrane was determined by measuring FRET between donor and acceptor. (B and C) Images of EGFR-WT (B) or EGFR-L858R (C) cells labeled with donor only (left), donor with acceptor (middle), and donor with acceptor in the presence of EGF (right). Shown are the masked intensity images restricted primarily to the plasma membrane (top) and donor fluorescence lifetime values, τ 2 , corresponding to the masked pixels (bottom). (D) Donor fluorescence lifetime values were averaged over many cells from five to six independent experiments for EGFR-WT and EGFR-L858R ± 2 μM NR12S acceptor and ± 30 nM EGF. Fluorescence lifetime results are summarized in Supplemental Table S2.

    Journal: Molecular Biology of the Cell

    Article Title: Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer

    doi: 10.1091/mbc.E15-05-0269

    Figure Lengend Snippet: FRET-FLIM reveals that unliganded EGFR-L858R adopts the extended conformation and requires ectodomain interactions for signaling. (A) Schematic of the EGFR ectodomain in the tethered, autoinhibited conformation (left) and the extended conformation (right). The donor fluorophore (Oregon Green 488, green) is covalently linked at the EGFR N-terminus via a small ACP tag, and the acceptor fluorophore (NR12S, a derivate of Nile Red, red) is embedded in the outer leaflet of the plasma membrane. The relative apparent separation between the EGFR N-terminus and the plasma membrane was determined by measuring FRET between donor and acceptor. (B and C) Images of EGFR-WT (B) or EGFR-L858R (C) cells labeled with donor only (left), donor with acceptor (middle), and donor with acceptor in the presence of EGF (right). Shown are the masked intensity images restricted primarily to the plasma membrane (top) and donor fluorescence lifetime values, τ 2 , corresponding to the masked pixels (bottom). (D) Donor fluorescence lifetime values were averaged over many cells from five to six independent experiments for EGFR-WT and EGFR-L858R ± 2 μM NR12S acceptor and ± 30 nM EGF. Fluorescence lifetime results are summarized in Supplemental Table S2.

    Article Snippet: The ACP-tagged EGFR-expressing CHO cells were plated and grown in four-well Lab-Tek chambered coverglass slides (Thermo Fisher) in DMEM with 10% fetal calf serum.

    Techniques: Labeling, Fluorescence

    Two-color dSTORM superresolution imaging shows ligand-induced EGFR dimerization and ligand-independent dimerization of EGFR mutants. (A) Representative two-color dSTORM-reconstructed image of a CHO cell expressing EGFR-WT in the presence of EGF, fixed and labeled with a monoclonal anti-EGFR antibody conjugated with either Alexa Fluor 647 (magenta) or Cy3B (green). Shown is a reconstructed superresolution image, in which each localization is represented as a two-dimensional Gaussian with σ proportional to its localization precision. The mean localization precisions for Alexa Fluor 647 and Cy3B are ∼10 and 12 nm, respectively. (B) Zoomed regions of A highlighting potential EGFR dimers (white boxes) separated by

    Journal: Molecular Biology of the Cell

    Article Title: Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer

    doi: 10.1091/mbc.E15-05-0269

    Figure Lengend Snippet: Two-color dSTORM superresolution imaging shows ligand-induced EGFR dimerization and ligand-independent dimerization of EGFR mutants. (A) Representative two-color dSTORM-reconstructed image of a CHO cell expressing EGFR-WT in the presence of EGF, fixed and labeled with a monoclonal anti-EGFR antibody conjugated with either Alexa Fluor 647 (magenta) or Cy3B (green). Shown is a reconstructed superresolution image, in which each localization is represented as a two-dimensional Gaussian with σ proportional to its localization precision. The mean localization precisions for Alexa Fluor 647 and Cy3B are ∼10 and 12 nm, respectively. (B) Zoomed regions of A highlighting potential EGFR dimers (white boxes) separated by

    Article Snippet: The ACP-tagged EGFR-expressing CHO cells were plated and grown in four-well Lab-Tek chambered coverglass slides (Thermo Fisher) in DMEM with 10% fetal calf serum.

    Techniques: Imaging, Expressing, Labeling

    EGFR mutations detected by three platforms using tumor tissue and plasma samples A heatmap representation of mutations detected from paired tumor tissue/pleural effusion and plasma samples, which were collected from 47 patients. The sample types and platforms used are shown in the left column. Forty-seven tumor tissue samples were detected using ARMS PCR, while 42 tumor samples and 6 pleural effusion samples were detected using Proton and the digital PCR platform. Patients with tumor tissue samples in ARMS PCR, but with pleural effusion samples in Proton and digital PCR were marked with the symbol “PE”. P42 had both tumor tissue and pleural effusion samples detected in Proton and digital PCR. Forty-seven paired plasma samples were detected using digital PCR only. The top two rows show patients’ gender and clinical stage, the upper middle three rows show tumor tissue samples tested by ARMS PCR, the middle six rows show tumor tissue or pleural effusion samples tested by Proton and digital PCR, and the bottom three rows show plasma samples tested by digital PCR only. Mutation events are represented by different colors, e.g., L858R by green, T790M by yellow, 19del by brown, and wild-type mutations by gray. The sites of ctDNA that were not detected are shown as blank.

    Journal: Oncotarget

    Article Title: Evaluation of digital PCR for detecting low-level EGFR mutations in advanced lung adenocarcinoma patients: a cross-platform comparison study

    doi: 10.18632/oncotarget.18866

    Figure Lengend Snippet: EGFR mutations detected by three platforms using tumor tissue and plasma samples A heatmap representation of mutations detected from paired tumor tissue/pleural effusion and plasma samples, which were collected from 47 patients. The sample types and platforms used are shown in the left column. Forty-seven tumor tissue samples were detected using ARMS PCR, while 42 tumor samples and 6 pleural effusion samples were detected using Proton and the digital PCR platform. Patients with tumor tissue samples in ARMS PCR, but with pleural effusion samples in Proton and digital PCR were marked with the symbol “PE”. P42 had both tumor tissue and pleural effusion samples detected in Proton and digital PCR. Forty-seven paired plasma samples were detected using digital PCR only. The top two rows show patients’ gender and clinical stage, the upper middle three rows show tumor tissue samples tested by ARMS PCR, the middle six rows show tumor tissue or pleural effusion samples tested by Proton and digital PCR, and the bottom three rows show plasma samples tested by digital PCR only. Mutation events are represented by different colors, e.g., L858R by green, T790M by yellow, 19del by brown, and wild-type mutations by gray. The sites of ctDNA that were not detected are shown as blank.

    Article Snippet: Detection of EGFR mutations by QuantStudio 3D Digital PCR Digital PCR assays were conducted using a QuantStudio 3D Digital PCR system (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Polymerase Chain Reaction, Digital PCR, Mutagenesis

    EGFR mutation frequency distribution of paired tumor tissue and plasma gDNA and ctDNA were detected in the digital PCR platform, and the frequency of tumor tissue is shown as a dot symbol, while the frequency of plasma is shown as a triangle symbol. Mutation types are shown by different colors. The frequency of plasma was lower than that of tumor tissue, and plasma did not detect a few positive sites in tumor tissue.

    Journal: Oncotarget

    Article Title: Evaluation of digital PCR for detecting low-level EGFR mutations in advanced lung adenocarcinoma patients: a cross-platform comparison study

    doi: 10.18632/oncotarget.18866

    Figure Lengend Snippet: EGFR mutation frequency distribution of paired tumor tissue and plasma gDNA and ctDNA were detected in the digital PCR platform, and the frequency of tumor tissue is shown as a dot symbol, while the frequency of plasma is shown as a triangle symbol. Mutation types are shown by different colors. The frequency of plasma was lower than that of tumor tissue, and plasma did not detect a few positive sites in tumor tissue.

    Article Snippet: Detection of EGFR mutations by QuantStudio 3D Digital PCR Digital PCR assays were conducted using a QuantStudio 3D Digital PCR system (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Mutagenesis, Digital PCR