egfr  (Millipore)

 
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    Name:
    Anti Epidermal Growth Factor antibody
    Description:
    Epidermal growth factor EGF is a growth factor that acts as mitogen for various types of cells of epithelial origin EGF binds to EGF receptor and induces tyrosine phosphorylation leading to proliferation of cells EGF R is present on many cell types of epithelial and mesenchymal lineages EGF R is capable of binding transforming growth factor α and heparin binding EGF in addition to EGF Some of the downstream pathways activated by EGF EGF R are PLCγ PI3K STAT3 PKC and MAPK cascades There are numerous effector molecule activated by EGF that result in a variety of biological processes such as morphogenesis cell motility apoptosis differentiation and organ repair and maintenance Deregulation of EGF R signaling is implicated in progression of a wide variety of tumors invasion and metastasis Anti Epidermal Growth Factor antibody is whole rabbit antiserum
    Catalog Number:
    E2635
    Price:
    None
    Applications:
    Anti-Epidermal Growth Factor antibody may be used to monitor EGF levels in organs, biological fluids, and cell culture media by means of radioimmunoassay (RIA), enzyme immunoassay (EIA) and immunohistological techniques. The antiserum may be used as a tool to determine purity when isolating EGF. Antibody dilution of 1:1000 to 1:5000 may be used for RIA. The antibody was used for immunohistochemistry of mouse salivary gland and liver sections at a working dilution of 1:100 and 1:1500, respectively. A dilution of 1:250 of this antibody was used to inject into female mice to study the process of embryo growth.
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    Structured Review

    Millipore egfr
    (A) Effect of IMC-A12 on <t>IGF-1R</t> and <t>EGFR</t> expression levels. Cells were exposed to IMC-A12and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (B). Effect of cetuximab and IMC-A12 on radiosensitivity of HNSCC lines in culture. Cells were treated with cetuximab and/or IMC-A12 and exposed to radiation as described under Methods. Survival curves were constructed with normalized values for the cytotoxicity induced by cetuximab/IMC-A12. Data shown are means ± SE from three independent experiments. RT only: black solid line; cetuximab + RT: green dash line; IMC-A12 + RT: blue dash line; cetuximab + IMC-A12 + RT: Red solid line.
    Epidermal growth factor EGF is a growth factor that acts as mitogen for various types of cells of epithelial origin EGF binds to EGF receptor and induces tyrosine phosphorylation leading to proliferation of cells EGF R is present on many cell types of epithelial and mesenchymal lineages EGF R is capable of binding transforming growth factor α and heparin binding EGF in addition to EGF Some of the downstream pathways activated by EGF EGF R are PLCγ PI3K STAT3 PKC and MAPK cascades There are numerous effector molecule activated by EGF that result in a variety of biological processes such as morphogenesis cell motility apoptosis differentiation and organ repair and maintenance Deregulation of EGF R signaling is implicated in progression of a wide variety of tumors invasion and metastasis Anti Epidermal Growth Factor antibody is whole rabbit antiserum
    https://www.bioz.com/result/egfr/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    egfr - by Bioz Stars, 2021-05
    93/100 stars

    Images

    1) Product Images from "Inhibition of EGFR or IGF-1R signaling enhances radiation response in head and neck cancer models but concurrent inhibition has no added benefit"

    Article Title: Inhibition of EGFR or IGF-1R signaling enhances radiation response in head and neck cancer models but concurrent inhibition has no added benefit

    Journal: Cancer Medicine

    doi: 10.1002/cam4.345

    (A) Effect of IMC-A12 on IGF-1R and EGFR expression levels. Cells were exposed to IMC-A12and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (B). Effect of cetuximab and IMC-A12 on radiosensitivity of HNSCC lines in culture. Cells were treated with cetuximab and/or IMC-A12 and exposed to radiation as described under Methods. Survival curves were constructed with normalized values for the cytotoxicity induced by cetuximab/IMC-A12. Data shown are means ± SE from three independent experiments. RT only: black solid line; cetuximab + RT: green dash line; IMC-A12 + RT: blue dash line; cetuximab + IMC-A12 + RT: Red solid line.
    Figure Legend Snippet: (A) Effect of IMC-A12 on IGF-1R and EGFR expression levels. Cells were exposed to IMC-A12and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (B). Effect of cetuximab and IMC-A12 on radiosensitivity of HNSCC lines in culture. Cells were treated with cetuximab and/or IMC-A12 and exposed to radiation as described under Methods. Survival curves were constructed with normalized values for the cytotoxicity induced by cetuximab/IMC-A12. Data shown are means ± SE from three independent experiments. RT only: black solid line; cetuximab + RT: green dash line; IMC-A12 + RT: blue dash line; cetuximab + IMC-A12 + RT: Red solid line.

    Techniques Used: Expressing, Western Blot, Construct

    (A) Effect of cetuximab on EGFR and IGF-1R expression levels. Cells were exposed to cetuximab and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (B) Effect of cetuximab on p-MAPK and p-Akt expression levels. Cells were exposed to cetuximab and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (C) Effect of cetuximab, radiation, or both on dimerization of EGFR and IGF1R. Cells were exposed to either cetuximab and/or 4 Gy and collected 10 min after irradiation. Whole cell lysates were subjected to immunoprecipitation (IP) with IGF-1R antibody and immunoblotted (IB) with EGFR antibody. Shown are representative Western blots of two independent experiments. Cont: Untreated control; cetux: cetuximab. p-MAPK: phosphorylated form of MAPK; p-AKT: phosphorylated form of Akt.
    Figure Legend Snippet: (A) Effect of cetuximab on EGFR and IGF-1R expression levels. Cells were exposed to cetuximab and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (B) Effect of cetuximab on p-MAPK and p-Akt expression levels. Cells were exposed to cetuximab and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (C) Effect of cetuximab, radiation, or both on dimerization of EGFR and IGF1R. Cells were exposed to either cetuximab and/or 4 Gy and collected 10 min after irradiation. Whole cell lysates were subjected to immunoprecipitation (IP) with IGF-1R antibody and immunoblotted (IB) with EGFR antibody. Shown are representative Western blots of two independent experiments. Cont: Untreated control; cetux: cetuximab. p-MAPK: phosphorylated form of MAPK; p-AKT: phosphorylated form of Akt.

    Techniques Used: Expressing, Western Blot, Irradiation, Immunoprecipitation

    2) Product Images from "Alternate Paths from Epidermal Growth Factor Receptor to Akt in Malignant Versus Nontransformed Lung Epithelial Cells"

    Article Title: Alternate Paths from Epidermal Growth Factor Receptor to Akt in Malignant Versus Nontransformed Lung Epithelial Cells

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2005-0049OC

    Complexes containing Shp2 with Gab1 and p85 in C10 and E10 cells. ( A and B ) C10/A5 and E10/E9 paired nontransformed/malignant cell lines were serum starved and treated with TGF-α with or without EGFR inhibitor PD153035. Lysates were immunoprecipitated
    Figure Legend Snippet: Complexes containing Shp2 with Gab1 and p85 in C10 and E10 cells. ( A and B ) C10/A5 and E10/E9 paired nontransformed/malignant cell lines were serum starved and treated with TGF-α with or without EGFR inhibitor PD153035. Lysates were immunoprecipitated

    Techniques Used: Immunoprecipitation

    3) Product Images from "Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration"

    Article Title: Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2014.209

    ErbB2 and EGFR are necessary for anoikis protection in KPL-4 and SUM149 cells, respectively. ( a ) KPL-4 cells were transduced with a lentivirus containing either an empty vector (EV) or an ErbB2 shRNA-containing vector (shErbB2), and western blotting analysis
    Figure Legend Snippet: ErbB2 and EGFR are necessary for anoikis protection in KPL-4 and SUM149 cells, respectively. ( a ) KPL-4 cells were transduced with a lentivirus containing either an empty vector (EV) or an ErbB2 shRNA-containing vector (shErbB2), and western blotting analysis

    Techniques Used: Transduction, Plasmid Preparation, shRNA, Western Blot

    4) Product Images from "Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration"

    Article Title: Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038825

    Representative immuno-fluorescent staining profiles for corneal epithelial phenotype. Corneal epithelial markers, including the differentiation markers, keratin 3 (K3), and connexin 43 (Cx43), as well as progenitor markers, EGFR, nuclear p63 and integrin β1, were expressed by HLE regenerated on human feeder of Hs68 fibroblasts; Hoechst 33342 was used for nuclear counterstaining.
    Figure Legend Snippet: Representative immuno-fluorescent staining profiles for corneal epithelial phenotype. Corneal epithelial markers, including the differentiation markers, keratin 3 (K3), and connexin 43 (Cx43), as well as progenitor markers, EGFR, nuclear p63 and integrin β1, were expressed by HLE regenerated on human feeder of Hs68 fibroblasts; Hoechst 33342 was used for nuclear counterstaining.

    Techniques Used: Staining

    5) Product Images from "Loss of Activating EGFR Mutant Gene Contributes to Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in Lung Cancer Cells"

    Article Title: Loss of Activating EGFR Mutant Gene Contributes to Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in Lung Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0041017

    The effect of erlotinib or knockdown of EGFR, HER2, or HER3 by their siRNAs on PC9/ER1 cells. A, Exponentially growing PC9 and PC9/ER1 cells were exposed to various doses of erlotinib for 5 h, and followed by western blot analysis. B, C, D, PC9 and PC9/ER1 cells were treated for 48 hr with 10 nM scramble (sc) siRNA, 2 nM or 10 nM EGFR siRNA, HER2 siRNA or HER3 siRNA respectively, and followed by Western blot analysis.
    Figure Legend Snippet: The effect of erlotinib or knockdown of EGFR, HER2, or HER3 by their siRNAs on PC9/ER1 cells. A, Exponentially growing PC9 and PC9/ER1 cells were exposed to various doses of erlotinib for 5 h, and followed by western blot analysis. B, C, D, PC9 and PC9/ER1 cells were treated for 48 hr with 10 nM scramble (sc) siRNA, 2 nM or 10 nM EGFR siRNA, HER2 siRNA or HER3 siRNA respectively, and followed by Western blot analysis.

    Techniques Used: Western Blot

    Our hypothetic model how drug resistance to erlotinib is acquired in lung cancer cells harboring activated mutant EGFR (mEGFR). Cell proliferation and survival of human lung cancer cells harboring activated mutant EGFR (PC9 and 11–18 cells) closely depend upon EGFR-driven PI3K/Akt pathway, and this proliferation/survival is highly susceptible to erlotinib and other EGFR TKIs. First, there is partial or complete loss of mEGFR gene allele in drug-resistant cell lines, and then gain of addiction to HER2/HER3 and PI3K/Akt signaling (PC9/ER1 cells). However, more definitive analysis on resistant cell lines of 11–18 is required because 11–18 resistant cell lines show only partial loss of mEGFR.
    Figure Legend Snippet: Our hypothetic model how drug resistance to erlotinib is acquired in lung cancer cells harboring activated mutant EGFR (mEGFR). Cell proliferation and survival of human lung cancer cells harboring activated mutant EGFR (PC9 and 11–18 cells) closely depend upon EGFR-driven PI3K/Akt pathway, and this proliferation/survival is highly susceptible to erlotinib and other EGFR TKIs. First, there is partial or complete loss of mEGFR gene allele in drug-resistant cell lines, and then gain of addiction to HER2/HER3 and PI3K/Akt signaling (PC9/ER1 cells). However, more definitive analysis on resistant cell lines of 11–18 is required because 11–18 resistant cell lines show only partial loss of mEGFR.

    Techniques Used: Mutagenesis

    Comparison of protein expression of EGFR family proteins and the down-stream molecules in erlotinib-resistant cell lines in the absence or presence of erlotinib. A, Western blot analysis showing the expression of pEGFR, EGFR, pHER2, HER2, pHER3, HER3, pc-Met, c-Met, PTEN, pAkt, Akt, pERK1/2, and ERK1/2 proteins, and α-tubulin as a loading control. B, Exponentially growing PC9 and PC9/ER1 cells were exposed to various doses of erlotinib for 5 hr, and followed by Western blot analysis. C, Exponentially growing 11–18, 11–18/ER1-7, and 11–18/ER2-1 cells were exposed to various doses of erlotinib for 5 hr, and followed by Western blot analysis.
    Figure Legend Snippet: Comparison of protein expression of EGFR family proteins and the down-stream molecules in erlotinib-resistant cell lines in the absence or presence of erlotinib. A, Western blot analysis showing the expression of pEGFR, EGFR, pHER2, HER2, pHER3, HER3, pc-Met, c-Met, PTEN, pAkt, Akt, pERK1/2, and ERK1/2 proteins, and α-tubulin as a loading control. B, Exponentially growing PC9 and PC9/ER1 cells were exposed to various doses of erlotinib for 5 hr, and followed by Western blot analysis. C, Exponentially growing 11–18, 11–18/ER1-7, and 11–18/ER2-1 cells were exposed to various doses of erlotinib for 5 hr, and followed by Western blot analysis.

    Techniques Used: Expressing, Western Blot

    6) Product Images from "Molecular characterization of novel trispecific ErbB-cMet-IGF1R antibodies and their antigen-binding properties"

    Article Title: Molecular characterization of novel trispecific ErbB-cMet-IGF1R antibodies and their antigen-binding properties

    Journal: Protein Engineering, Design and Selection

    doi: 10.1093/protein/gzs048

    ( a ) Overlay of SPR sensorgrams showing simultaneous binding of EGFR, IGF1R and Her3 (plus cMet as negative control) to TriMAb2. TriMAb2 was captured onto the sensor chip and binding of antigens studied by four consecutive injects (with a technical lack phase after second inject) of soluble receptors, permutating the order in different runs. Each run is highlighted by a different color. ( b ) Heat of receptor binding to MAbs measured by ITC and fitted to a 1 : 1 binding event curve. Top panel: soluble receptors titrated into a solution of their corresponding parental MAb in three independent experiments. Bottom panel: the three receptors titrated one after the other into the same solution of TriMab2. The consecutive titrations are evaluated and depicted separately. A colour version of this figure is available as supplementary data at PEDS online.
    Figure Legend Snippet: ( a ) Overlay of SPR sensorgrams showing simultaneous binding of EGFR, IGF1R and Her3 (plus cMet as negative control) to TriMAb2. TriMAb2 was captured onto the sensor chip and binding of antigens studied by four consecutive injects (with a technical lack phase after second inject) of soluble receptors, permutating the order in different runs. Each run is highlighted by a different color. ( b ) Heat of receptor binding to MAbs measured by ITC and fitted to a 1 : 1 binding event curve. Top panel: soluble receptors titrated into a solution of their corresponding parental MAb in three independent experiments. Bottom panel: the three receptors titrated one after the other into the same solution of TriMab2. The consecutive titrations are evaluated and depicted separately. A colour version of this figure is available as supplementary data at PEDS online.

    Techniques Used: SPR Assay, Binding Assay, Negative Control, Chromatin Immunoprecipitation

    7) Product Images from "IGFBP2 potentiates nuclear EGFR-STAT3 signaling"

    Article Title: IGFBP2 potentiates nuclear EGFR-STAT3 signaling

    Journal: Oncogene

    doi: 10.1038/onc.2015.131

    IGFBP2 correlates with STAT3 activation and nuclear EGFR localization in clinical samples Expression and localization of IGFBP2, pSTAT3(Y705) and EGFR were detected with immunohistochemistry from a TMA that included 222 human grade 2-4 gliomas. ( A ) TMA immunostaining images (magnification 40×) representing weak and strong staining of IGFBP2 and pSTAT3(Y705). ( B ) Cytosolic and nuclear IGFBP2 expression associated with the percentage of cells positive for pSTAT3 and with pSTAT3 staining intensity. Bar graphs illustrate the increasing fractions of pSTAT3-positive cells and pSTAT3 intensity upon increasing IGFBP2 intensity or nuclear accumulation. ( C ) TMA immunostaining images (magnification 40×) representing low and high nuclear localization of IGFBP2 and EGFR. ( D ) Nuclear IGFBP2 associated with nuclear EGFR. The bar graph illustrates the fraction of samples with increasing nuclear EGFR localization upon increasing nuclear accumulation of IGFBP2. ( E ) Nuclear co-localization of IGFBP2, EGFR and phosphorylated STAT3 predicted poor survival among patients with human grade 2-4 glioma. Patients were stratified into 2 cohorts based on the nuclear staining of all 3 proteins: triple positives (≥1% of cells with nuclear expression, n=51, red line) and all other cases (n=83, blue line). Survival rates were visualized by using a Kaplan-Meier survival plot (p=0.0086).
    Figure Legend Snippet: IGFBP2 correlates with STAT3 activation and nuclear EGFR localization in clinical samples Expression and localization of IGFBP2, pSTAT3(Y705) and EGFR were detected with immunohistochemistry from a TMA that included 222 human grade 2-4 gliomas. ( A ) TMA immunostaining images (magnification 40×) representing weak and strong staining of IGFBP2 and pSTAT3(Y705). ( B ) Cytosolic and nuclear IGFBP2 expression associated with the percentage of cells positive for pSTAT3 and with pSTAT3 staining intensity. Bar graphs illustrate the increasing fractions of pSTAT3-positive cells and pSTAT3 intensity upon increasing IGFBP2 intensity or nuclear accumulation. ( C ) TMA immunostaining images (magnification 40×) representing low and high nuclear localization of IGFBP2 and EGFR. ( D ) Nuclear IGFBP2 associated with nuclear EGFR. The bar graph illustrates the fraction of samples with increasing nuclear EGFR localization upon increasing nuclear accumulation of IGFBP2. ( E ) Nuclear co-localization of IGFBP2, EGFR and phosphorylated STAT3 predicted poor survival among patients with human grade 2-4 glioma. Patients were stratified into 2 cohorts based on the nuclear staining of all 3 proteins: triple positives (≥1% of cells with nuclear expression, n=51, red line) and all other cases (n=83, blue line). Survival rates were visualized by using a Kaplan-Meier survival plot (p=0.0086).

    Techniques Used: Activation Assay, Expressing, Immunohistochemistry, Immunostaining, Staining

    IGFBP2 activates STAT3 through EGFR ( A) Immunoblot analysis of SNB19 cells starved of serum overnight then stimulated with exogenous IGFBP2 protein at the indicated dosages (0, 50, 100, 250 ng/mL) for 60 minutes. Densitometric analysis shown below the immunoblot indicates fold-change relative to unstimulated control cells (normalized to beta-actin loading control or total protein for phosphorylated proteins). ( B ) Immunoblot analysis of U87 cells starved of serum overnight then stimulated with exogenous IGFBP2 (100ng/mL) for the indicated time points (0, 5, 10, 15, 30, 60 minutes). Densitometric analysis shown below the immunoblot indicates fold-change relative to unstimulated control cells (normalized to loading control or total protein for phosphorylated proteins). ( C ) Immunoblot analysis comparing stable SNB19 empty vector cells (SNB19.EV) to SNB19 cells stably overexpressing IGFBP2 (SNB19.BP2). Densitometric analysis shown below the immunoblot indicates fold-change relative to SNB19.EV after normalization to beta-tubulin loading control (or total protein for phosphorylated proteins). ( D ) Immunoblot analysis comparing SNB19.EV and SNB19.BP2 cells depleted of EGFR via 2 independent pools of EGFR siRNA (EGFR sir#1, EGFR sir#2) to cells transfected with scrambled negative control siRNA (ctrl siR). The intensity of pSTAT3(Y705), quantified by densitometry, is shown below the immunoblot as fold-change relative to control siRNA, normalized to total STAT3.
    Figure Legend Snippet: IGFBP2 activates STAT3 through EGFR ( A) Immunoblot analysis of SNB19 cells starved of serum overnight then stimulated with exogenous IGFBP2 protein at the indicated dosages (0, 50, 100, 250 ng/mL) for 60 minutes. Densitometric analysis shown below the immunoblot indicates fold-change relative to unstimulated control cells (normalized to beta-actin loading control or total protein for phosphorylated proteins). ( B ) Immunoblot analysis of U87 cells starved of serum overnight then stimulated with exogenous IGFBP2 (100ng/mL) for the indicated time points (0, 5, 10, 15, 30, 60 minutes). Densitometric analysis shown below the immunoblot indicates fold-change relative to unstimulated control cells (normalized to loading control or total protein for phosphorylated proteins). ( C ) Immunoblot analysis comparing stable SNB19 empty vector cells (SNB19.EV) to SNB19 cells stably overexpressing IGFBP2 (SNB19.BP2). Densitometric analysis shown below the immunoblot indicates fold-change relative to SNB19.EV after normalization to beta-tubulin loading control (or total protein for phosphorylated proteins). ( D ) Immunoblot analysis comparing SNB19.EV and SNB19.BP2 cells depleted of EGFR via 2 independent pools of EGFR siRNA (EGFR sir#1, EGFR sir#2) to cells transfected with scrambled negative control siRNA (ctrl siR). The intensity of pSTAT3(Y705), quantified by densitometry, is shown below the immunoblot as fold-change relative to control siRNA, normalized to total STAT3.

    Techniques Used: Plasmid Preparation, Stable Transfection, Transfection, Negative Control

    IGFBP2 co-precipitates and co-localizes with EGFR ( A ) Co-immunoprecipitation (IP) of IGFBP2 and EGFR in SNB19.EV control cells versus SNB19.BP2 cells analyzed by immunoblot (IB). ( B ) Immunoprecipitation of IGFBP2 in U87 cells starved of serum overnight then stimulated with 2 different doses of IGFBP2 for 30 minutes, analyzed by immunoblotting. ( C ) Confocal microscopy images of immunofluorescence staining for IGFBP2 (green), EGFR (red) and DAPI (blue) in SNB19.BP2 cells show IGFBP2 and EGFR co-localization; blue arrow = cell membrane; purple arrow = cytoplasm; white arrow = nucleus.
    Figure Legend Snippet: IGFBP2 co-precipitates and co-localizes with EGFR ( A ) Co-immunoprecipitation (IP) of IGFBP2 and EGFR in SNB19.EV control cells versus SNB19.BP2 cells analyzed by immunoblot (IB). ( B ) Immunoprecipitation of IGFBP2 in U87 cells starved of serum overnight then stimulated with 2 different doses of IGFBP2 for 30 minutes, analyzed by immunoblotting. ( C ) Confocal microscopy images of immunofluorescence staining for IGFBP2 (green), EGFR (red) and DAPI (blue) in SNB19.BP2 cells show IGFBP2 and EGFR co-localization; blue arrow = cell membrane; purple arrow = cytoplasm; white arrow = nucleus.

    Techniques Used: Immunoprecipitation, Confocal Microscopy, Immunofluorescence, Staining

    IGFBP2 drives EGFR nuclear accumulation ( A ) Immunoblot analysis of cytoplasmic (cyt) and nuclear (nuc) fractions of SNB19.EV and SNB19.BP2 cells. Beta-tubulin represents a loading control for the cytoplasmic fraction, and PARP represents a loading control for the nuclear fraction. Densitometric analysis represented by the bar graph, demonstrates percentage of cytoplasmic or nuclear EGFR. ( B ) Confocal images of SNB19 parental cells and SNB19 parental cells stimulated with exogenous IGFBP2 protein (250ng/mL for 30 minutes). Cells were stained for EGFR (red) and the nuclei stained with DAPI (blue). ( C ) Immunoblot analysis of cytoplasmic and nuclear fractions of SNB19 parental cells stimulated with exogenous IGFBP2 (250ng/mL for indicated times). The graph represents fold-change of cytoplasmic or nuclear IGFBP2 and EGFR calculated from densitometric analysis of the immunoblot bands. ( D) Immunoblot analysis comparing cytoplasmic and nuclear fractions of SNB19.BP2 cells depleted of IGFBP2 via 2 independent pools of IGFBP2 siRNA (BP2 siR #1, #2) to cells transfected with scrambled negative control siRNA (ctrl siR). Densitometric analysis represented by the bar graph, demonstrates percentage of cytoplasmic or nuclear EGFR. ( E ) Immunoblot analysis of cytoplasmic and nuclear fractions of transiently transfected SNB19.EV, SNB19.BP2 wild type (BP2 WT) and SNB19 with a mutated IGFBP2 nuclear localization signal (BP2ΔNLS). Densitometric analysis represented by the bar graph, demonstrates percentage of cytoplasmic or nuclear EGFR. ( F ) Immunoblot analysis of cytoplasmic and nuclear proteins in stable SNB19.EV, SNB19.BP2 WT and SNB19.BP2ΔNLS cells.
    Figure Legend Snippet: IGFBP2 drives EGFR nuclear accumulation ( A ) Immunoblot analysis of cytoplasmic (cyt) and nuclear (nuc) fractions of SNB19.EV and SNB19.BP2 cells. Beta-tubulin represents a loading control for the cytoplasmic fraction, and PARP represents a loading control for the nuclear fraction. Densitometric analysis represented by the bar graph, demonstrates percentage of cytoplasmic or nuclear EGFR. ( B ) Confocal images of SNB19 parental cells and SNB19 parental cells stimulated with exogenous IGFBP2 protein (250ng/mL for 30 minutes). Cells were stained for EGFR (red) and the nuclei stained with DAPI (blue). ( C ) Immunoblot analysis of cytoplasmic and nuclear fractions of SNB19 parental cells stimulated with exogenous IGFBP2 (250ng/mL for indicated times). The graph represents fold-change of cytoplasmic or nuclear IGFBP2 and EGFR calculated from densitometric analysis of the immunoblot bands. ( D) Immunoblot analysis comparing cytoplasmic and nuclear fractions of SNB19.BP2 cells depleted of IGFBP2 via 2 independent pools of IGFBP2 siRNA (BP2 siR #1, #2) to cells transfected with scrambled negative control siRNA (ctrl siR). Densitometric analysis represented by the bar graph, demonstrates percentage of cytoplasmic or nuclear EGFR. ( E ) Immunoblot analysis of cytoplasmic and nuclear fractions of transiently transfected SNB19.EV, SNB19.BP2 wild type (BP2 WT) and SNB19 with a mutated IGFBP2 nuclear localization signal (BP2ΔNLS). Densitometric analysis represented by the bar graph, demonstrates percentage of cytoplasmic or nuclear EGFR. ( F ) Immunoblot analysis of cytoplasmic and nuclear proteins in stable SNB19.EV, SNB19.BP2 WT and SNB19.BP2ΔNLS cells.

    Techniques Used: Staining, Transfection, Negative Control

    8) Product Images from "89Zr-Onartuzumab PET imaging of c-MET receptor dynamics"

    Article Title: 89Zr-Onartuzumab PET imaging of c-MET receptor dynamics

    Journal: European Journal of Nuclear Medicine and Molecular Imaging

    doi: 10.1007/s00259-017-3672-x

    a In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827ErlRes cells normalized to expression in parental cell line HCC827. b In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of erlotinib for 4 days. c In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827 and HCC827ErlRes cells after 24 h treatment with 25, 50 and 100 nM NVP-AUY-922 normalized to untreated controls. d In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of NVP-AUY-922 for 4 days
    Figure Legend Snippet: a In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827ErlRes cells normalized to expression in parental cell line HCC827. b In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of erlotinib for 4 days. c In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827 and HCC827ErlRes cells after 24 h treatment with 25, 50 and 100 nM NVP-AUY-922 normalized to untreated controls. d In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of NVP-AUY-922 for 4 days

    Techniques Used: In Vitro, Flow Cytometry, Expressing, MTT Assay, Proliferation Assay

    9) Product Images from "Heparin Binding Epidermal Growth Factor-Like Growth Factor and PD169316 Prevent Apoptosis in Mouse Embryonic Stem Cells"

    Article Title: Heparin Binding Epidermal Growth Factor-Like Growth Factor and PD169316 Prevent Apoptosis in Mouse Embryonic Stem Cells

    Journal:

    doi: 10.1093/jb/mvn153

    Identification of EGFR phosphorylation sites involved in anti-CD9-induced apoptosis (20×). Twenty-four hrs of anti-CD9 antibody treatment was performed, cells were fixed and stained for activated EGFR Y845, 1068, 1086, 1148 and 1173 (green). Nuclei
    Figure Legend Snippet: Identification of EGFR phosphorylation sites involved in anti-CD9-induced apoptosis (20×). Twenty-four hrs of anti-CD9 antibody treatment was performed, cells were fixed and stained for activated EGFR Y845, 1068, 1086, 1148 and 1173 (green). Nuclei

    Techniques Used: Staining

    Phosphorylation of EGFR is suppressed by HB-EGF and p38 MAPK inhibitor (20×). After 24 h of anti-CD9 antibody treatment and fixation cells were stained for activated EGFR 1148 and 1173 (green). Nuclei are labelled with DAPI. Anti-CD9 antibody
    Figure Legend Snippet: Phosphorylation of EGFR is suppressed by HB-EGF and p38 MAPK inhibitor (20×). After 24 h of anti-CD9 antibody treatment and fixation cells were stained for activated EGFR 1148 and 1173 (green). Nuclei are labelled with DAPI. Anti-CD9 antibody

    Techniques Used: Staining

    10) Product Images from "Expression and Pharmacological Inhibition of TrkB and EGFR in Glioblastoma"

    Article Title: Expression and Pharmacological Inhibition of TrkB and EGFR in Glioblastoma

    Journal: bioRxiv

    doi: 10.1101/2020.02.03.932608

    Inhibition of TrkB and EGFR alone or in combination reduces human GBM cell viability. Time course analysis of cell viability, by trypan blue cell counting, were performed after 24, 48 and 72 h of exposure to ANA-12 or AG 1478 exposure in A172 (A, B) and U87MG (C, D) cells. Dose-response curves were evaluated by trypan blue cell counting after treatment with increasing concentrations of ANA-12 (1-50μM) or AG 1478 (1-30 μM) for 48 h in A172 cells (E, F) and 24 h in U87MG cells (G, H). The drug vehicles (DMSO or EtOH) served as controls. Dose-response curves after combined treatment with ANA-12 and AG 1478 were evaluated after 48 h of drug exposure in A172 cells (I) and 24 h in U87MG cells (J). Data are expressed by mean ± SEM and represent three independent experiments * p
    Figure Legend Snippet: Inhibition of TrkB and EGFR alone or in combination reduces human GBM cell viability. Time course analysis of cell viability, by trypan blue cell counting, were performed after 24, 48 and 72 h of exposure to ANA-12 or AG 1478 exposure in A172 (A, B) and U87MG (C, D) cells. Dose-response curves were evaluated by trypan blue cell counting after treatment with increasing concentrations of ANA-12 (1-50μM) or AG 1478 (1-30 μM) for 48 h in A172 cells (E, F) and 24 h in U87MG cells (G, H). The drug vehicles (DMSO or EtOH) served as controls. Dose-response curves after combined treatment with ANA-12 and AG 1478 were evaluated after 48 h of drug exposure in A172 cells (I) and 24 h in U87MG cells (J). Data are expressed by mean ± SEM and represent three independent experiments * p

    Techniques Used: Inhibition, Cell Counting

    Synergistic effect after combined inhibition of TrkB and EGFR in A172 GBM cells. A172 and U87MG cell lines were treated with varying concentrations of ANA-12 and AG 1478 alone or in combination. The IC50-values were calculated from the dose-response curves after different exposure times (48 h for A172 and 24 h for U87MG cells) and expressed with their respective 95% confidence intervals and summarized in the table (A). The combination index (CI) was determined by the method of Chou-Talalay and data are presented as mean ± SEM (B).
    Figure Legend Snippet: Synergistic effect after combined inhibition of TrkB and EGFR in A172 GBM cells. A172 and U87MG cell lines were treated with varying concentrations of ANA-12 and AG 1478 alone or in combination. The IC50-values were calculated from the dose-response curves after different exposure times (48 h for A172 and 24 h for U87MG cells) and expressed with their respective 95% confidence intervals and summarized in the table (A). The combination index (CI) was determined by the method of Chou-Talalay and data are presented as mean ± SEM (B).

    Techniques Used: Inhibition

    Inhibition of TrkB and EGFR in an intracranial GBM mouse model. (A) A total of 40,000 U87MG cells were intracranially (i.c.) injected into nude mice. Drug treatments started on the seventh day after cell implantation. The animals were randomly divided in 4 groups ( n= 5 per group) to receive intraperitoneal (i.p.) injections for 21 days, and were treated by a blinded investigator with ANA-12 (1 mg/kg daily plus vehicle every 3 days), AG1478 (10 mg/kg every three days plus vehicle daily), ANA-12 (1 mg/kg daily) plus AG 1478 (10 mg/kg/every three days) and vehicle (DMSO) daily. After 21 days of treatment, the animals received an i.p. injection of 5-ALA (50 mg/kg) and after 1 h mice were euthanized by cervical dislocation and the brains were removed to be analyzed (B) Representative IVIS images of harvested brains acquired at day 29 after transplantation. (C) Corresponding data of 5-ALA (PPIX) radiant efficiency from the whole brain. (D) After performing images of the intact brain, a brain matrix was used to cut sequential 1-mm slices through the region containing the tumor. Slices were imaged using IVIS and fluorescence images were collected. Data are expressed as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post-hoc tests. No statistical differences were observed between experimental groups.
    Figure Legend Snippet: Inhibition of TrkB and EGFR in an intracranial GBM mouse model. (A) A total of 40,000 U87MG cells were intracranially (i.c.) injected into nude mice. Drug treatments started on the seventh day after cell implantation. The animals were randomly divided in 4 groups ( n= 5 per group) to receive intraperitoneal (i.p.) injections for 21 days, and were treated by a blinded investigator with ANA-12 (1 mg/kg daily plus vehicle every 3 days), AG1478 (10 mg/kg every three days plus vehicle daily), ANA-12 (1 mg/kg daily) plus AG 1478 (10 mg/kg/every three days) and vehicle (DMSO) daily. After 21 days of treatment, the animals received an i.p. injection of 5-ALA (50 mg/kg) and after 1 h mice were euthanized by cervical dislocation and the brains were removed to be analyzed (B) Representative IVIS images of harvested brains acquired at day 29 after transplantation. (C) Corresponding data of 5-ALA (PPIX) radiant efficiency from the whole brain. (D) After performing images of the intact brain, a brain matrix was used to cut sequential 1-mm slices through the region containing the tumor. Slices were imaged using IVIS and fluorescence images were collected. Data are expressed as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post-hoc tests. No statistical differences were observed between experimental groups.

    Techniques Used: Inhibition, Injection, Mouse Assay, Transplantation Assay, Fluorescence

    Inhibition of TrkB and EGFR alone or in combination in a subcutaneous GBM xenograft mouse model. U87MG cells were pretreated in vitro for 24 h with ANA-12 (13.85 μM), AG-1478 (13.26 μM) or ANA-12 plus AG-1478, and the viable cells were injected into the flanks of nude mice (6-7 mice per group) as shown in the schematic drawing (A). Caliper measurements were used to determine the displayed subcutaneous tumor volume. Mice were euthanized and tumors were excised when volume reached approximately 800-1,000 mm 3 (B) Tumors were measured every 2 days and volumes were calculated as described in materials and methods section. Tumor growth is represented by tumor volume (mm 3 ) at the indicated days; Control ( n = 7), ANA-12 ( n= 8), AG 1478 ( n= 8) and ANA-12 plus AG 1478 ( n= 8) (C) Tumor growth curve is shown on selected time points of 15, 31 and 45 days to highlight statistical differences (D). Tumor volumes (mm 3 ) at the time of tissue harvest. (E) Tumor weight (mg) at the time of tissue harvest are shown in (F) Kaplan-Meier curves presenting percent of mice surviving following tumor implantation. Data are expressed as mean ± SEM (* p
    Figure Legend Snippet: Inhibition of TrkB and EGFR alone or in combination in a subcutaneous GBM xenograft mouse model. U87MG cells were pretreated in vitro for 24 h with ANA-12 (13.85 μM), AG-1478 (13.26 μM) or ANA-12 plus AG-1478, and the viable cells were injected into the flanks of nude mice (6-7 mice per group) as shown in the schematic drawing (A). Caliper measurements were used to determine the displayed subcutaneous tumor volume. Mice were euthanized and tumors were excised when volume reached approximately 800-1,000 mm 3 (B) Tumors were measured every 2 days and volumes were calculated as described in materials and methods section. Tumor growth is represented by tumor volume (mm 3 ) at the indicated days; Control ( n = 7), ANA-12 ( n= 8), AG 1478 ( n= 8) and ANA-12 plus AG 1478 ( n= 8) (C) Tumor growth curve is shown on selected time points of 15, 31 and 45 days to highlight statistical differences (D). Tumor volumes (mm 3 ) at the time of tissue harvest. (E) Tumor weight (mg) at the time of tissue harvest are shown in (F) Kaplan-Meier curves presenting percent of mice surviving following tumor implantation. Data are expressed as mean ± SEM (* p

    Techniques Used: Inhibition, In Vitro, Injection, Mouse Assay, Tumor Implantation

    11) Product Images from "Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein-ubiquitin conjugates"

    Article Title: Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein-ubiquitin conjugates

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200112080

    TSG101 and hVPS28 are localized to ubiquitin-rich EGF-positive endosomes. (A) A431 cells were starved overnight in serum-free medium and either left untreated (no EGF) or incubated with 0.4 μg/ml EGF at 4°C followed by the indicated time at 37°C. Cell lysates were immunoprecipitated with control (left) or anti-EGFR antibody (right). Bound fractions were Western blotted with anti-EGFR (top) or antiubiquitin (FK2; bottom). The band detected in the FK2 blot running just below the EGFR is an artifact, since it is found in EGFR immunoprecipitates from cells lacking EGFR (unpublished data). (B) Oregon green EGF was bound to A431 cells and internalized as indicated. Cells were chilled, incubated with saponin before fixation, and then analyzed for EGF content (left) and stained with FK2 antibody (right). (C) A431 cells were either left untreated (left) or had Oregon green EGF internalized for 60 min (right). Cells were chilled and incubated with saponin before fixation and then analyzed for EGF (green) or stained (red) for the following markers: EEA1, polyubiquitin–protein conjugates (FK1), TSG101, or hVPS28.
    Figure Legend Snippet: TSG101 and hVPS28 are localized to ubiquitin-rich EGF-positive endosomes. (A) A431 cells were starved overnight in serum-free medium and either left untreated (no EGF) or incubated with 0.4 μg/ml EGF at 4°C followed by the indicated time at 37°C. Cell lysates were immunoprecipitated with control (left) or anti-EGFR antibody (right). Bound fractions were Western blotted with anti-EGFR (top) or antiubiquitin (FK2; bottom). The band detected in the FK2 blot running just below the EGFR is an artifact, since it is found in EGFR immunoprecipitates from cells lacking EGFR (unpublished data). (B) Oregon green EGF was bound to A431 cells and internalized as indicated. Cells were chilled, incubated with saponin before fixation, and then analyzed for EGF content (left) and stained with FK2 antibody (right). (C) A431 cells were either left untreated (left) or had Oregon green EGF internalized for 60 min (right). Cells were chilled and incubated with saponin before fixation and then analyzed for EGF (green) or stained (red) for the following markers: EEA1, polyubiquitin–protein conjugates (FK1), TSG101, or hVPS28.

    Techniques Used: Incubation, Immunoprecipitation, Western Blot, Staining

    Inhibition of proteasome function inhibits EGF degradation and prevents deubiquitination of endosomal proteins. (A, left) 125 I EGF was bound and internalized into A431 cells in the absence (control; C) or presence (M) of 5 μM MG132 or with MG132 in cells which had also been preincubated for 3 h with 5 μM MG132 (P). After the indicated times of internalization, medium was removed and assayed for degraded 125 I EGF. (A, right) 125 I EGF was bound and internalized into untreated A431 cells (C) or into cells to which MG132 was added 30 min after internalization had begun (M). Samples were assayed for EGF degradation as above. All values are means of triplicate determinations ± SEM. (B) A431 Cells were incubated with 0.4 μg/ml EGF at 4°C followed by 10, 30, or 120 min at 37°C as indicated. Cells were incubated with 5 μM MG132 for 3 h before and during internalization (P), incubated with 5 μM MG132 during internalization only (M), or otherwise untreated (C). Cell lysates were immunoprecipitated with anti-EGFR antibody, and bound fractions were Western blotted with anti-EGFR (top) or antiubiquitin (FK2; bottom). The samples containing peak FK2 labeling are indicated by asterisks. An immunoprecipitate from untreated cells is also shown (U). (C) Oregon green EGF was bound to A431 cells and internalized. Cells were chilled, incubated with saponin before fixation, and then analyzed for EGF content (green) and stained with FK2 antibody (red). (Top) Cells preincubated for 3 h with MG132 and then EGF internalized for 1 h with MG132. (Middle) EGF internalized for 1 h with MG132. (Bottom) EGF internalized for 30 min followed by a further 4.5 h with MG132.
    Figure Legend Snippet: Inhibition of proteasome function inhibits EGF degradation and prevents deubiquitination of endosomal proteins. (A, left) 125 I EGF was bound and internalized into A431 cells in the absence (control; C) or presence (M) of 5 μM MG132 or with MG132 in cells which had also been preincubated for 3 h with 5 μM MG132 (P). After the indicated times of internalization, medium was removed and assayed for degraded 125 I EGF. (A, right) 125 I EGF was bound and internalized into untreated A431 cells (C) or into cells to which MG132 was added 30 min after internalization had begun (M). Samples were assayed for EGF degradation as above. All values are means of triplicate determinations ± SEM. (B) A431 Cells were incubated with 0.4 μg/ml EGF at 4°C followed by 10, 30, or 120 min at 37°C as indicated. Cells were incubated with 5 μM MG132 for 3 h before and during internalization (P), incubated with 5 μM MG132 during internalization only (M), or otherwise untreated (C). Cell lysates were immunoprecipitated with anti-EGFR antibody, and bound fractions were Western blotted with anti-EGFR (top) or antiubiquitin (FK2; bottom). The samples containing peak FK2 labeling are indicated by asterisks. An immunoprecipitate from untreated cells is also shown (U). (C) Oregon green EGF was bound to A431 cells and internalized. Cells were chilled, incubated with saponin before fixation, and then analyzed for EGF content (green) and stained with FK2 antibody (red). (Top) Cells preincubated for 3 h with MG132 and then EGF internalized for 1 h with MG132. (Middle) EGF internalized for 1 h with MG132. (Bottom) EGF internalized for 30 min followed by a further 4.5 h with MG132.

    Techniques Used: Inhibition, Incubation, Immunoprecipitation, Western Blot, Labeling, Staining

    Depletion of TSG101 affects EGF trafficking and endosomal ubiquitination. (A) HeLa cells were mock transfected (lane 1) or transfected with siRNA against Tsg101 (lane 2). Cell extracts were Western blotted for TSG101 (top) or tubulin as a control (bottom). (B) HeLa cells were mock transfected (M; top) or transfected with siRNA against Tsg101 (T; bottom) and stained for EEA1 or CD63 as indicated. (C). Oregon green EGF was internalized for the indicated times into mock-transfected HeLa cells (top) or cells transfected with siRNA against Tsg101 (bottom). Cells were analyzed for EGF (green) and stained with DAPI (blue) and with FK2 monoclonal antibody followed by Texas red 2° antibody (red). (D). EGF was bound and internalized into mock-transfected HeLa cells (left) or cells transfected with siRNA against Tsg101 (right). Cell lysates were made after the indicated times of EGF internalization and Western blotted for EGFR.
    Figure Legend Snippet: Depletion of TSG101 affects EGF trafficking and endosomal ubiquitination. (A) HeLa cells were mock transfected (lane 1) or transfected with siRNA against Tsg101 (lane 2). Cell extracts were Western blotted for TSG101 (top) or tubulin as a control (bottom). (B) HeLa cells were mock transfected (M; top) or transfected with siRNA against Tsg101 (T; bottom) and stained for EEA1 or CD63 as indicated. (C). Oregon green EGF was internalized for the indicated times into mock-transfected HeLa cells (top) or cells transfected with siRNA against Tsg101 (bottom). Cells were analyzed for EGF (green) and stained with DAPI (blue) and with FK2 monoclonal antibody followed by Texas red 2° antibody (red). (D). EGF was bound and internalized into mock-transfected HeLa cells (left) or cells transfected with siRNA against Tsg101 (right). Cell lysates were made after the indicated times of EGF internalization and Western blotted for EGFR.

    Techniques Used: Transfection, Western Blot, Staining

    12) Product Images from "Constitutive and ligand-induced EGFR signaling triggers distinct and mutually exclusive downstream signaling networks"

    Article Title: Constitutive and ligand-induced EGFR signaling triggers distinct and mutually exclusive downstream signaling networks

    Journal: Nature communications

    doi: 10.1038/ncomms6811

    EGFR forms a signaling complex with IRF3 and TBK1 (a) EGFR co-immunoprecipitates with IRF3 in U251EGFR cells in the absence of EGF. When EGF is added there is a rapid loss of the EGFR-IRF3 association. (b) Similarly, the EGFR coimmunoprecipitates with TBK1 in U251EGFR cells. When EGF is added there is a rapid loss of the EGFR-TBK1 association. (c) TBK1 is associated with IRF3 in U251EGFR cells in the absence of EGF. When EGF is added to cells, there is a rapid decrease in TBK-IRF3 association. The TBK1-IRF3 association is also disrupted when Erlotinib is used suggesting that it is EGFR driven. C refers to control vehicle (PBS). (d) EGFR co-immunoprecipitates with IRF3 in MDAMB468 cells in the absence of EGF. When EGF is added there is a rapid loss of the EGFR-IRF3 association. (e) Similarly, the EGFR coimmunoprecipitates with TBK1 in MDAMB468 cells. When EGF is added there is a rapid loss of the EGFR-TBK1 association. (f) TBK1 is associated with IRF3 in MDAMB468 cells in the absence of EGF. When EGF is added to cells, there is a rapid decrease in TBK-IRF3 association. The TBK1-IRF3 association is also disrupted when Erlotinib is used suggesting that it is EGFR driven. (g) In contradistinction to IRF3 and TBK1, Shc becomes associated with the EGFR only when EGF is added to U251EGFR cells. (h) About 10% of IRF3 is associated with the EGFR. (i) About 5% of phospho-IRF3 is associated with the EGFR. (j) EGFR associates with IRF3 and TBK1 in the membrane fraction. Membrane, cytosolic or nuclear fractions were prepared from U251EGFR cells. Immunoprecipitation was conducted with IRF3 or TBK1 antibody followed by Western blot with EGFR. E-Cadherin, GAPDH and lamin blots were done to test purity of various fractions. Cells were serum starved and cultured in serum free DMEM overnight for all experiments. The EGF concentration used was 50ng/ml. (k) In vitro transcribed and translated EGFR was incubated with GST-IRF3 or GST alone followed by Western blot with EGFR. (l) shows in vitro transcribed and translated EGFR. V: empty vector.
    Figure Legend Snippet: EGFR forms a signaling complex with IRF3 and TBK1 (a) EGFR co-immunoprecipitates with IRF3 in U251EGFR cells in the absence of EGF. When EGF is added there is a rapid loss of the EGFR-IRF3 association. (b) Similarly, the EGFR coimmunoprecipitates with TBK1 in U251EGFR cells. When EGF is added there is a rapid loss of the EGFR-TBK1 association. (c) TBK1 is associated with IRF3 in U251EGFR cells in the absence of EGF. When EGF is added to cells, there is a rapid decrease in TBK-IRF3 association. The TBK1-IRF3 association is also disrupted when Erlotinib is used suggesting that it is EGFR driven. C refers to control vehicle (PBS). (d) EGFR co-immunoprecipitates with IRF3 in MDAMB468 cells in the absence of EGF. When EGF is added there is a rapid loss of the EGFR-IRF3 association. (e) Similarly, the EGFR coimmunoprecipitates with TBK1 in MDAMB468 cells. When EGF is added there is a rapid loss of the EGFR-TBK1 association. (f) TBK1 is associated with IRF3 in MDAMB468 cells in the absence of EGF. When EGF is added to cells, there is a rapid decrease in TBK-IRF3 association. The TBK1-IRF3 association is also disrupted when Erlotinib is used suggesting that it is EGFR driven. (g) In contradistinction to IRF3 and TBK1, Shc becomes associated with the EGFR only when EGF is added to U251EGFR cells. (h) About 10% of IRF3 is associated with the EGFR. (i) About 5% of phospho-IRF3 is associated with the EGFR. (j) EGFR associates with IRF3 and TBK1 in the membrane fraction. Membrane, cytosolic or nuclear fractions were prepared from U251EGFR cells. Immunoprecipitation was conducted with IRF3 or TBK1 antibody followed by Western blot with EGFR. E-Cadherin, GAPDH and lamin blots were done to test purity of various fractions. Cells were serum starved and cultured in serum free DMEM overnight for all experiments. The EGF concentration used was 50ng/ml. (k) In vitro transcribed and translated EGFR was incubated with GST-IRF3 or GST alone followed by Western blot with EGFR. (l) shows in vitro transcribed and translated EGFR. V: empty vector.

    Techniques Used: Immunoprecipitation, Western Blot, Cell Culture, Concentration Assay, In Vitro, Incubation, Plasmid Preparation

    13) Product Images from "89Zr-Onartuzumab PET imaging of c-MET receptor dynamics"

    Article Title: 89Zr-Onartuzumab PET imaging of c-MET receptor dynamics

    Journal: European Journal of Nuclear Medicine and Molecular Imaging

    doi: 10.1007/s00259-017-3672-x

    a In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827ErlRes cells normalized to expression in parental cell line HCC827. b In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of erlotinib for 4 days. c In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827 and HCC827ErlRes cells after 24 h treatment with 25, 50 and 100 nM NVP-AUY-922 normalized to untreated controls. d In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of NVP-AUY-922 for 4 days
    Figure Legend Snippet: a In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827ErlRes cells normalized to expression in parental cell line HCC827. b In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of erlotinib for 4 days. c In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827 and HCC827ErlRes cells after 24 h treatment with 25, 50 and 100 nM NVP-AUY-922 normalized to untreated controls. d In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of NVP-AUY-922 for 4 days

    Techniques Used: In Vitro, Flow Cytometry, Expressing, MTT Assay, Proliferation Assay

    14) Product Images from "EGFR and HER2 Activate Rigidity Sensing Only on Rigid Matrices"

    Article Title: EGFR and HER2 Activate Rigidity Sensing Only on Rigid Matrices

    Journal: Nature materials

    doi: 10.1038/nmat4893

    EGFR and HER2 affect local contractility through Src. a , Cell area with respect to time. Cells were pre-incubated with PP2 (200 nM) for 30 min prior to plating on FN-coated pillar substrates in serum-free medium. n > 15 cells, n > 5 independent experiments. b, Average of number of CUs per 10 min monitored for 7 hours. c , Area of Cos-7 cells expressing myosin IIA when transfected with siRNA for EGFR and mutations of EGFR or HER2 on FN coated stiff pillars. d , Average number of CUs in 10 minute windows for the cells shown in c. Error bars show standard error of the mean.
    Figure Legend Snippet: EGFR and HER2 affect local contractility through Src. a , Cell area with respect to time. Cells were pre-incubated with PP2 (200 nM) for 30 min prior to plating on FN-coated pillar substrates in serum-free medium. n > 15 cells, n > 5 independent experiments. b, Average of number of CUs per 10 min monitored for 7 hours. c , Area of Cos-7 cells expressing myosin IIA when transfected with siRNA for EGFR and mutations of EGFR or HER2 on FN coated stiff pillars. d , Average number of CUs in 10 minute windows for the cells shown in c. Error bars show standard error of the mean.

    Techniques Used: Incubation, Expressing, Transfection

    15) Product Images from "Adhesion-Mediated Squamous Cell Carcinoma Survival through Ligand-Independent Activation of Epidermal Growth Factor Receptor"

    Article Title: Adhesion-Mediated Squamous Cell Carcinoma Survival through Ligand-Independent Activation of Epidermal Growth Factor Receptor

    Journal: The American Journal of Pathology

    doi:

    Cell-cell adhesion-induced EGFR activation suppresses anoikis. A: DNA fragmentation of HSC-3 cells. HSC-3 cells were plated as MCA culture in the absence or presence of 1 μg/ml AG1478 for 48 hours ( lanes 2 and 3 ). Suspended single cells (SC) alone ( lane 4 ) and monolayers (ML) cultured for 48 hours in the presence of 1 μmol/L AG1478 ( lane 5 ) were used as controls. The DNA laddering assay for intranucleosomal DNA cleavage was performed as described in Experimental Procedures. Lane 1 : Standard 100-bp DNA ladder. B: TUNEL analysis of AG1478-treated HSC-3 cells. HSC-3 cells were plated as ML, MCA, or SC culture for 48 hours before TUNEL analysis. AG1478 (1 μmol/L) was added to the culture as indicated. Values represent the apoptotic cell fractions (%) that stained positive with FITC-dUTP.
    Figure Legend Snippet: Cell-cell adhesion-induced EGFR activation suppresses anoikis. A: DNA fragmentation of HSC-3 cells. HSC-3 cells were plated as MCA culture in the absence or presence of 1 μg/ml AG1478 for 48 hours ( lanes 2 and 3 ). Suspended single cells (SC) alone ( lane 4 ) and monolayers (ML) cultured for 48 hours in the presence of 1 μmol/L AG1478 ( lane 5 ) were used as controls. The DNA laddering assay for intranucleosomal DNA cleavage was performed as described in Experimental Procedures. Lane 1 : Standard 100-bp DNA ladder. B: TUNEL analysis of AG1478-treated HSC-3 cells. HSC-3 cells were plated as ML, MCA, or SC culture for 48 hours before TUNEL analysis. AG1478 (1 μmol/L) was added to the culture as indicated. Values represent the apoptotic cell fractions (%) that stained positive with FITC-dUTP.

    Techniques Used: Activation Assay, Cell Culture, DNA Laddering, TUNEL Assay, Staining

    E-cadherin-mediated cell-cell adhesion induces EGFR activation. A: HSC-3 cells were cultured as MCAs for 0.5 hours ( a ), 3 hours ( b , c ), or 6 hours ( d , e ) and then transferred onto glass coverslips and immunolabeled for EGFR, E-cadherin, or activated EGFR (p-EGFR) as indicated. In C , which represents the early stage of cell aggregation, the top frame stained for E-cadherin is of a different cell pair than the others. Specimens were analyzed by confocal microscopy, and representative images are shown. B: Immunoblot analysis of activated EGFR during MCA formation. As controls, HSC-3 cells were cultured in suspension as single cells (SCs) for 24 hours or treated with 1 μmol/L tyrphostin AG1478 for 24 hours in MCA culture. Relative protein densities of phosphorylated EGFR were determined; EGFR kinase activity is expressed as fold induction relative to the HSC-3 MCA at 0 hours. Data reported here are representative of four independent experiments. C: Effect of anti-E-cadherin mAb on HSC-3 MCA formation. HSC-3 cells were plated as MCA culture in the absence or presence of 50 μg/ml anti-E-cadherin mAb HECD-1 for 24 hours. D: HSC-3 cells were plated as MCA culture in the presence of 50 μg/ml anti-E-cadherin mAb HECD-1 or mouse IgG for 24 hours. Equivalent protein was immunoblotted with mAbs to phospho-EGFR and total EGFR.
    Figure Legend Snippet: E-cadherin-mediated cell-cell adhesion induces EGFR activation. A: HSC-3 cells were cultured as MCAs for 0.5 hours ( a ), 3 hours ( b , c ), or 6 hours ( d , e ) and then transferred onto glass coverslips and immunolabeled for EGFR, E-cadherin, or activated EGFR (p-EGFR) as indicated. In C , which represents the early stage of cell aggregation, the top frame stained for E-cadherin is of a different cell pair than the others. Specimens were analyzed by confocal microscopy, and representative images are shown. B: Immunoblot analysis of activated EGFR during MCA formation. As controls, HSC-3 cells were cultured in suspension as single cells (SCs) for 24 hours or treated with 1 μmol/L tyrphostin AG1478 for 24 hours in MCA culture. Relative protein densities of phosphorylated EGFR were determined; EGFR kinase activity is expressed as fold induction relative to the HSC-3 MCA at 0 hours. Data reported here are representative of four independent experiments. C: Effect of anti-E-cadherin mAb on HSC-3 MCA formation. HSC-3 cells were plated as MCA culture in the absence or presence of 50 μg/ml anti-E-cadherin mAb HECD-1 for 24 hours. D: HSC-3 cells were plated as MCA culture in the presence of 50 μg/ml anti-E-cadherin mAb HECD-1 or mouse IgG for 24 hours. Equivalent protein was immunoblotted with mAbs to phospho-EGFR and total EGFR.

    Techniques Used: Activation Assay, Cell Culture, Immunolabeling, Staining, Confocal Microscopy, Activity Assay

    Assembly and co-localization of the EGFR/E-cadherin complex is independent of EGFR kinase activity. A: HSC-3 cells were cultured as MCAs for 6 hours in the absence or presence of AG1478 (1 μmol/L) and then transferred onto glass coverslips and immunolabeled for EGFR (green) and E-cadherin or activated EGFR (p-EGFR) (red). Specimens were analyzed by confocal microscopy, and representative images are shown. B: HSC-3 cells were plated as MCA for 12 hours in the absence or presence of AG1478 (1 μmol/L). E-cadherin was immunoprecipitated (IP) from total cell lysates with HECD-1 mAb. The presence of activated EGFR (p-EGFR) or total EGFR in the immunoprecipitate was detected with specific anti-p-EGFR or anti-EGFR mAb.
    Figure Legend Snippet: Assembly and co-localization of the EGFR/E-cadherin complex is independent of EGFR kinase activity. A: HSC-3 cells were cultured as MCAs for 6 hours in the absence or presence of AG1478 (1 μmol/L) and then transferred onto glass coverslips and immunolabeled for EGFR (green) and E-cadherin or activated EGFR (p-EGFR) (red). Specimens were analyzed by confocal microscopy, and representative images are shown. B: HSC-3 cells were plated as MCA for 12 hours in the absence or presence of AG1478 (1 μmol/L). E-cadherin was immunoprecipitated (IP) from total cell lysates with HECD-1 mAb. The presence of activated EGFR (p-EGFR) or total EGFR in the immunoprecipitate was detected with specific anti-p-EGFR or anti-EGFR mAb.

    Techniques Used: Activity Assay, Cell Culture, Immunolabeling, Confocal Microscopy, Immunoprecipitation

    16) Product Images from "Dacomitinib, a pan-inhibitor of ErbB receptors, suppresses growth and invasive capacity of chemoresistant ovarian carcinoma cells"

    Article Title: Dacomitinib, a pan-inhibitor of ErbB receptors, suppresses growth and invasive capacity of chemoresistant ovarian carcinoma cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04147-0

    The ErbB family contributes to cisplatin resistance. ( A ) Correlation of expression of HRG1 - α , HRG1 - β , EGFR and HER2 with resistance to cisplatin. EOC cell lines with higher expression of HRG1 - α , HRG1 - β , EGFR and HER2 showed significantly higher cisplatin IC 50 values. The correlation coefficient (r) between the expression of EGFR and HER2 and cisplatin concentrations was 0.917 ( P = 0.0281) and 0.890 ( P = 0.0341), respectively. ( B ) Dacomitinib inhibits ErbB activation. The effect of dacomitinib (5 μM) on ErbB activation was determined by Western blot analysis. Protein lysates were subjected to Western blotting and probed with the indicated antibodies. β-actin was used as the loading control. The blots are representative of three independent experiments with similar results. ( C ) The effects of the ErbB inhibitors on HRGβ-1-induced proliferation in cisplatin-treated Caov4 cells were shown by MTT assay. The cells were pre-treated with the anti-ErbB agents for 4 h, followed by treatment with HRGβ-1 for 48 h. ( D ) The effects of the ErbB inhibitors-cisplatin therapy on cell proliferation were investigated by MTT assay after 48 h of treatment and the data are shown by IC 50 shift analysis. The concentrations of cisplatin were 0.1, 0.5, 1, 2.5, 5 and 10 μg/mL. ( E ) Normalised isobolograms of combination of erlotinib (5 μM) and dacomitinib (5 μM) with cisplatin. The data were analysed using the CalcuSyn software. The connecting line represents additivity. Data points located below the line indicate a synergistic drug-drug interaction and data points above the line indicate an antagonistic interaction. The numbers under the isobolograms indicate the concentrations of the drugs in combination. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p
    Figure Legend Snippet: The ErbB family contributes to cisplatin resistance. ( A ) Correlation of expression of HRG1 - α , HRG1 - β , EGFR and HER2 with resistance to cisplatin. EOC cell lines with higher expression of HRG1 - α , HRG1 - β , EGFR and HER2 showed significantly higher cisplatin IC 50 values. The correlation coefficient (r) between the expression of EGFR and HER2 and cisplatin concentrations was 0.917 ( P = 0.0281) and 0.890 ( P = 0.0341), respectively. ( B ) Dacomitinib inhibits ErbB activation. The effect of dacomitinib (5 μM) on ErbB activation was determined by Western blot analysis. Protein lysates were subjected to Western blotting and probed with the indicated antibodies. β-actin was used as the loading control. The blots are representative of three independent experiments with similar results. ( C ) The effects of the ErbB inhibitors on HRGβ-1-induced proliferation in cisplatin-treated Caov4 cells were shown by MTT assay. The cells were pre-treated with the anti-ErbB agents for 4 h, followed by treatment with HRGβ-1 for 48 h. ( D ) The effects of the ErbB inhibitors-cisplatin therapy on cell proliferation were investigated by MTT assay after 48 h of treatment and the data are shown by IC 50 shift analysis. The concentrations of cisplatin were 0.1, 0.5, 1, 2.5, 5 and 10 μg/mL. ( E ) Normalised isobolograms of combination of erlotinib (5 μM) and dacomitinib (5 μM) with cisplatin. The data were analysed using the CalcuSyn software. The connecting line represents additivity. Data points located below the line indicate a synergistic drug-drug interaction and data points above the line indicate an antagonistic interaction. The numbers under the isobolograms indicate the concentrations of the drugs in combination. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p

    Techniques Used: Expressing, Activation Assay, Western Blot, MTT Assay, Software

    17) Product Images from "EGFR is Essential for TLR3 Signaling"

    Article Title: EGFR is Essential for TLR3 Signaling

    Journal: Science signaling

    doi: 10.1126/scisignal.2002581

    Src interaction with TLR3 is independent of TRIF and TRAF3, but dependent on EGFR
    Figure Legend Snippet: Src interaction with TLR3 is independent of TRIF and TRAF3, but dependent on EGFR

    Techniques Used:

    Src and EGFR are required for phosphorylation of Tyr 759 and Tyr 858 in TLR3
    Figure Legend Snippet: Src and EGFR are required for phosphorylation of Tyr 759 and Tyr 858 in TLR3

    Techniques Used:

    The TIR domain of TLR3 is not required for its interaction with Src and EGFR
    Figure Legend Snippet: The TIR domain of TLR3 is not required for its interaction with Src and EGFR

    Techniques Used:

    18) Product Images from "Tamoxifen Induces Cytotoxic Autophagy in Glioblastoma"

    Article Title: Tamoxifen Induces Cytotoxic Autophagy in Glioblastoma

    Journal: Journal of Neuropathology and Experimental Neurology

    doi: 10.1093/jnen/nlw071

    Inhibition of protein kinase C (PKC) recapitulates the effects of OHT on EGFR. (A) PKC inhibitors Ro31 and trifluoperazine (TFP) increase LC3-II accumulation and reduce EGFR protein levels after 24 hours as determined by Western blot. (B) Phosphorylation
    Figure Legend Snippet: Inhibition of protein kinase C (PKC) recapitulates the effects of OHT on EGFR. (A) PKC inhibitors Ro31 and trifluoperazine (TFP) increase LC3-II accumulation and reduce EGFR protein levels after 24 hours as determined by Western blot. (B) Phosphorylation

    Techniques Used: Inhibition, Western Blot

    OHT accelerates degradation of epidermal growth factor receptor (EGFR). (A) Treatment with OHT (24 hours) results in a concentration-dependent decrease in EGFR protein levels in U87 but not U87vIII cells. (B) U87 cells possessing EGFRvIII (U87vIII)
    Figure Legend Snippet: OHT accelerates degradation of epidermal growth factor receptor (EGFR). (A) Treatment with OHT (24 hours) results in a concentration-dependent decrease in EGFR protein levels in U87 but not U87vIII cells. (B) U87 cells possessing EGFRvIII (U87vIII)

    Techniques Used: Concentration Assay

    19) Product Images from "Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells, et al. Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells"

    Article Title: Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells, et al. Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14730

    Expression level of EGFR and MET in generated variants of A375 and WM9 melanoma cell lines. A, Results of qRT‐PCR analysis of EGFR and MET expression are shown as the mean (relative expression compared to GAPDH) ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001. B, Western blotting analysis of EGFR and MET protein level in generated cell lines. Membranes were probed with antibodies directed against total EGFR and MET, and are representative for at least three independent experiments. GAPDH was used as the sample loading control
    Figure Legend Snippet: Expression level of EGFR and MET in generated variants of A375 and WM9 melanoma cell lines. A, Results of qRT‐PCR analysis of EGFR and MET expression are shown as the mean (relative expression compared to GAPDH) ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001. B, Western blotting analysis of EGFR and MET protein level in generated cell lines. Membranes were probed with antibodies directed against total EGFR and MET, and are representative for at least three independent experiments. GAPDH was used as the sample loading control

    Techniques Used: Expressing, Generated, Quantitative RT-PCR, Western Blot

    Proteolytic activity of melanoma cells with altered EGFR and MET expression. A, Representative pictures of proteolytic activity of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells (F‐actin visible in red) detected using FITC‐conjugated gelatin (green). Gelatin degradation indicated with white arrows is visualized as the dark areas on the fluorescently labelled gelatin background. Scale bar—10 µm. B, Quantification of digestion area calculated using ImageJ software from at least 40 cells from three independent experiments. Results are presented as the mean ± SD. C, MMP‐9 activity in concentrated conditioned media tested by gelatin zymography with (D) densitometric analysis. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Proteolytic activity of melanoma cells with altered EGFR and MET expression. A, Representative pictures of proteolytic activity of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells (F‐actin visible in red) detected using FITC‐conjugated gelatin (green). Gelatin degradation indicated with white arrows is visualized as the dark areas on the fluorescently labelled gelatin background. Scale bar—10 µm. B, Quantification of digestion area calculated using ImageJ software from at least 40 cells from three independent experiments. Results are presented as the mean ± SD. C, MMP‐9 activity in concentrated conditioned media tested by gelatin zymography with (D) densitometric analysis. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Activity Assay, Expressing, Software, Zymography

    Impact of EGFR and MET on invasion abilities and invadopodia formation in examined melanoma cell lines. A, The invasion assay performed on transwell filters coated with Matrigel for 24 h. Relative invasion factor was calculated versus control cells, where number of invading control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. B, Representative pictures of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells seeded on Matrigel‐coated coverslips stained for F‐actin (red), cortactin (green) and cell nuclei (blue). Arrows indicate invadopodia. Scale bar—8 μm. C, Quantification of the average number of invadopodia in examined cells. Invadopodia formed by at least 40 cells from three independent experiments were counted, and results are presented as the mean ± SD. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Impact of EGFR and MET on invasion abilities and invadopodia formation in examined melanoma cell lines. A, The invasion assay performed on transwell filters coated with Matrigel for 24 h. Relative invasion factor was calculated versus control cells, where number of invading control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. B, Representative pictures of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells seeded on Matrigel‐coated coverslips stained for F‐actin (red), cortactin (green) and cell nuclei (blue). Arrows indicate invadopodia. Scale bar—8 μm. C, Quantification of the average number of invadopodia in examined cells. Invadopodia formed by at least 40 cells from three independent experiments were counted, and results are presented as the mean ± SD. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Invasion Assay, Staining

    Migration abilities of melanoma cells with altered level of EGFR and MET. A, Cell trajectories and (B) migration distances of single A375 and WM9 cells analysed for 48 h using IncuCyte ® Live‐Cell Analysis System and ImageJ software. C, Representative images of wound closure, which was (D) quantified as per cent of area colonized by cells within 48 h (based on pictures analysed with an IncuCyte ® Scratch Wound Cell Migration Software Module). E, The migration assay executed on transwell filters for 24 h. Relative migration factor was calculated versus control cells, where number of migrating control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Migration abilities of melanoma cells with altered level of EGFR and MET. A, Cell trajectories and (B) migration distances of single A375 and WM9 cells analysed for 48 h using IncuCyte ® Live‐Cell Analysis System and ImageJ software. C, Representative images of wound closure, which was (D) quantified as per cent of area colonized by cells within 48 h (based on pictures analysed with an IncuCyte ® Scratch Wound Cell Migration Software Module). E, The migration assay executed on transwell filters for 24 h. Relative migration factor was calculated versus control cells, where number of migrating control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Migration, Software

    20) Product Images from "Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells, et al. Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells"

    Article Title: Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells, et al. Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14730

    Expression level of EGFR and MET in generated variants of A375 and WM9 melanoma cell lines. A, Results of qRT‐PCR analysis of EGFR and MET expression are shown as the mean (relative expression compared to GAPDH) ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001. B, Western blotting analysis of EGFR and MET protein level in generated cell lines. Membranes were probed with antibodies directed against total EGFR and MET, and are representative for at least three independent experiments. GAPDH was used as the sample loading control
    Figure Legend Snippet: Expression level of EGFR and MET in generated variants of A375 and WM9 melanoma cell lines. A, Results of qRT‐PCR analysis of EGFR and MET expression are shown as the mean (relative expression compared to GAPDH) ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001. B, Western blotting analysis of EGFR and MET protein level in generated cell lines. Membranes were probed with antibodies directed against total EGFR and MET, and are representative for at least three independent experiments. GAPDH was used as the sample loading control

    Techniques Used: Expressing, Generated, Quantitative RT-PCR, Western Blot

    Proteolytic activity of melanoma cells with altered EGFR and MET expression. A, Representative pictures of proteolytic activity of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells (F‐actin visible in red) detected using FITC‐conjugated gelatin (green). Gelatin degradation indicated with white arrows is visualized as the dark areas on the fluorescently labelled gelatin background. Scale bar—10 µm. B, Quantification of digestion area calculated using ImageJ software from at least 40 cells from three independent experiments. Results are presented as the mean ± SD. C, MMP‐9 activity in concentrated conditioned media tested by gelatin zymography with (D) densitometric analysis. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Proteolytic activity of melanoma cells with altered EGFR and MET expression. A, Representative pictures of proteolytic activity of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells (F‐actin visible in red) detected using FITC‐conjugated gelatin (green). Gelatin degradation indicated with white arrows is visualized as the dark areas on the fluorescently labelled gelatin background. Scale bar—10 µm. B, Quantification of digestion area calculated using ImageJ software from at least 40 cells from three independent experiments. Results are presented as the mean ± SD. C, MMP‐9 activity in concentrated conditioned media tested by gelatin zymography with (D) densitometric analysis. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Activity Assay, Expressing, Software, Zymography

    Impact of EGFR and MET on invasion abilities and invadopodia formation in examined melanoma cell lines. A, The invasion assay performed on transwell filters coated with Matrigel for 24 h. Relative invasion factor was calculated versus control cells, where number of invading control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. B, Representative pictures of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells seeded on Matrigel‐coated coverslips stained for F‐actin (red), cortactin (green) and cell nuclei (blue). Arrows indicate invadopodia. Scale bar—8 μm. C, Quantification of the average number of invadopodia in examined cells. Invadopodia formed by at least 40 cells from three independent experiments were counted, and results are presented as the mean ± SD. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Impact of EGFR and MET on invasion abilities and invadopodia formation in examined melanoma cell lines. A, The invasion assay performed on transwell filters coated with Matrigel for 24 h. Relative invasion factor was calculated versus control cells, where number of invading control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. B, Representative pictures of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells seeded on Matrigel‐coated coverslips stained for F‐actin (red), cortactin (green) and cell nuclei (blue). Arrows indicate invadopodia. Scale bar—8 μm. C, Quantification of the average number of invadopodia in examined cells. Invadopodia formed by at least 40 cells from three independent experiments were counted, and results are presented as the mean ± SD. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Invasion Assay, Staining

    Migration abilities of melanoma cells with altered level of EGFR and MET. A, Cell trajectories and (B) migration distances of single A375 and WM9 cells analysed for 48 h using IncuCyte ® Live‐Cell Analysis System and ImageJ software. C, Representative images of wound closure, which was (D) quantified as per cent of area colonized by cells within 48 h (based on pictures analysed with an IncuCyte ® Scratch Wound Cell Migration Software Module). E, The migration assay executed on transwell filters for 24 h. Relative migration factor was calculated versus control cells, where number of migrating control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Migration abilities of melanoma cells with altered level of EGFR and MET. A, Cell trajectories and (B) migration distances of single A375 and WM9 cells analysed for 48 h using IncuCyte ® Live‐Cell Analysis System and ImageJ software. C, Representative images of wound closure, which was (D) quantified as per cent of area colonized by cells within 48 h (based on pictures analysed with an IncuCyte ® Scratch Wound Cell Migration Software Module). E, The migration assay executed on transwell filters for 24 h. Relative migration factor was calculated versus control cells, where number of migrating control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Migration, Software

    21) Product Images from "Access to the Nucleus and Functional Association with c-Myc Is Required for the Full Oncogenic Potential of ?EGFR/EGFRvIII *"

    Article Title: Access to the Nucleus and Functional Association with c-Myc Is Required for the Full Oncogenic Potential of ?EGFR/EGFRvIII *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.399352

    ΔEGFR contains NES and NLS signals that are functional. A , schematic of EGFR depicting the deleted region of ΔEGFR as well as the location of the NLS and NES signals. SS , signal peptide; ECD , extracellular domain; TM , transmembrane domain; KD , kinase domain; CR , C-terminal regulatory region. B , confocal analysis of GFP-ΔEGFR and its mutants ( green ) in U87 cells. C , stable expression of V5-ΔEGFR and the mutants in U87 cells. D . E , confocal analyses of U87 stable cells using V5 antibody ( green ). Nuclei were counterstained using DAPI ( blue ) for all confocal analyses. F , coimmunoprecipitation ( IP ) and Western blot analysis ( WB .
    Figure Legend Snippet: ΔEGFR contains NES and NLS signals that are functional. A , schematic of EGFR depicting the deleted region of ΔEGFR as well as the location of the NLS and NES signals. SS , signal peptide; ECD , extracellular domain; TM , transmembrane domain; KD , kinase domain; CR , C-terminal regulatory region. B , confocal analysis of GFP-ΔEGFR and its mutants ( green ) in U87 cells. C , stable expression of V5-ΔEGFR and the mutants in U87 cells. D . E , confocal analyses of U87 stable cells using V5 antibody ( green ). Nuclei were counterstained using DAPI ( blue ) for all confocal analyses. F , coimmunoprecipitation ( IP ) and Western blot analysis ( WB .

    Techniques Used: Functional Assay, Expressing, Western Blot

    Related Articles

    Immunofluorescence:

    Article Title: Estrogen Receptor ? Exerts Tumor Repressive Functions in Human Malignant Pleural Mesothelioma via EGFR Inactivation and Affects Response to Gefitinib
    Article Snippet: The monoclonal antibody specific for Akt and the phosphorylation site-specific polyclonal antibodies for ERK1 (pThr202 and pTyr204), ERK2 (pThr185 and pTyr187) MAP kinases, and Akt (pSer473 and pThr308) were from Cell Signaling Technology (Beverly, MA). .. The polyclonal antibodies for EGFR and ERβ, used in immunofluorescence analysis were obtained from Calbiochem (Darmstadt, Germany) and Zymed-Invitrogen (Carlsbad, CA), respectively. .. Protein A-Sepharose and ECL were from Amersham Pharmacia Biotech (Uppsala, Sweden).

    Labeling:

    Article Title: Polysaccharides from Ganoderma lucidum Promote Cognitive Function and Neural Progenitor Proliferation in Mouse Model of Alzheimer's Disease
    Article Snippet: Proteins in the cell lysates were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. .. Proteins were labeled with primary antibodies as follows: rabbit monoclonal antibodies against phospo-FGFR1 (Tyr653/Tyr654, 06-1433, Millipore), EGFR (06-847, Millipore), FGFR1 (9740, Cell Signaling Technology), phospho-EGFR (Tyr1068, 2234, Cell Signaling), phospho-ERK (9101, Cell Signaling), ERK (9102, Cell Signaling), phospho-AKT (4060, Cell Signaling) or AKT (4691, Cell Signaling), BDNF (sc-546, Santa Cruz Biotechnology), and ACTIN (A2066, Sigma-Aldrich). .. Immunoreactive bands on the membrane were revealed by chemiluminescent detection (Bio-Rad) of peroxidase-conjugated, subtype-specific antibody (M21002, Abmart).

    Western Blot:

    Article Title: Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma
    Article Snippet: The TF/chromatin-associated DNA was purified and quantified by qPCR using primers spanning TEAD1--accessible open chromatin regions in multiple genes, and normalized to input (Supplementary Table for primer sequences). .. Western immunoblot and immunofluorescenceImmunoblotting was performed by standard protocol using the following antibodies: TEAD1 (ThermoFisher, PA5-37075, 1:100, and BD, 610923, 1:100), TEAD4 (Santa Cruz sc-101184, 1:500), EGFR (Millipore, 06–847, 1:1000), pEGFR (Abcam, ab40815, 1:2000), AQP4 (Novus, NBP187679m 1:500), ERK (Santa Cruz, sc-135900, 1:500), pERK (Santa Cruz, sc-81492, 1:500), AKT (Cell Signaling, 4691T, 1:500), pAKT (Cell Signaling, 4060T, 1:500), CDH11 (ThermoFisher, 71–7600, 1:250) and ACTB (ThermoFisher, MA5-15739, 1:10,000), which were diluted in 2.5% BSA in TBS-T solution. .. HRP-linked anti-mouse (GE Healthcare, NA9311ML) and anti-rabbit (GE Healthcare, NA9341ML) secondary antibodies were used (1:500) to detect immunoreactivity.

    Article Title: DDX3X Induces Primary EGFR-TKI Resistance Based on Intratumor Heterogeneity in Lung Cancer Cells Harboring EGFR-Activating Mutations
    Article Snippet: Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on Mini-PROTEAN TGX Any kD Precast Gels (BioRad, Hercules, CA, USA) and subsequently transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). .. Immunoblots from tumor cell lysates were probed with antibodies against DDX3X (Sigma), EGFR, phospho-EGFR (Tyr1068), phospho-EGFR (Tyr1173), phospho-EGFR (Tyr845), Akt, phospho-Akt, Erk1/2, phospho-Erk1/2, and β-actin (Sigma). .. All antibodies except for anti-DDX3X and anti-β-actin were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA).

    Article Title: PML mediates glioblastoma resistance to mammalian target of rapamycin (mTOR)-targeted therapies
    Article Snippet: .. We used antibodies directed against the following: phosopho-Akt Ser473, Akt, phospho-S6 Ser235/236, S6, phospho-Erk, Erk, CyclinD1, cleaved PARP (Cell Signaling); β-actin, p21 (Sigma); phospho-EGFR Tyr1086 (Invitrogen); EGFR (Millipore); PML (for Western blotting, Abcam; for immunohistochemisry, Santa Cruz). .. Reagents used are rapamycin, As2 O3 , polybrene (Sigma), erlotinib (ChemieTex), pp242 (Chemdea).

    Binding Assay:

    Article Title: ATP-mediated Activation of the NADPH Oxidase DUOX1 Mediates Airway Epithelial Responses to Bacterial Stimuli *
    Article Snippet: HBE1 cells were starved for 24 h and stimulated with either ATP or α-ASGM1 for 2 h, and conditioned media were collected for analysis of TGF-α protein levels using ELISA (BD Biosciences). .. To avoid TGF-α binding to EGFR, cells were in some cases pretreated with an α-EGFR mAb (225; 4 μg/ml; Calbiochem) for 30 min prior to stimulation. .. Upon starvation, cell monolayers were stimulated in the presence of 10 μ m of fluorogenic ADAM17 Substrate II (Calbiochem) for 2 h, after which media were collected.

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    Millipore a549 cells
    SPOCK2 inhibits influenza virus infection. (A) <t>A549</t> cells were transfected with V5-tagged SPOCK2 or empty vector. After 24 h, the cells were infected with PR8 IAV (MOIs, 0.01, 0.1, and 1) for the indicated times. HA vRNA levels were measured by qRT-PCR. Expression was normalized to GAPDH. In the graph, HA expression at 6 h in the EMPTY-V5-transfected sample was set to 1 at each dose. The graph presents the average values from triplicate experiments. Error bars represent SDs. *, P
    A549 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The Phospho EGFR pTyr1068 ELISA Enzyme Linked Immunosorbent Assay kit is a very rapid convenient and sensitive assay kit that can monitor the activation or function of important biological pathways
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    Image Search Results


    SPOCK2 inhibits influenza virus infection. (A) A549 cells were transfected with V5-tagged SPOCK2 or empty vector. After 24 h, the cells were infected with PR8 IAV (MOIs, 0.01, 0.1, and 1) for the indicated times. HA vRNA levels were measured by qRT-PCR. Expression was normalized to GAPDH. In the graph, HA expression at 6 h in the EMPTY-V5-transfected sample was set to 1 at each dose. The graph presents the average values from triplicate experiments. Error bars represent SDs. *, P

    Journal: Journal of Virology

    Article Title: The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells

    doi: 10.1128/JVI.00662-19

    Figure Lengend Snippet: SPOCK2 inhibits influenza virus infection. (A) A549 cells were transfected with V5-tagged SPOCK2 or empty vector. After 24 h, the cells were infected with PR8 IAV (MOIs, 0.01, 0.1, and 1) for the indicated times. HA vRNA levels were measured by qRT-PCR. Expression was normalized to GAPDH. In the graph, HA expression at 6 h in the EMPTY-V5-transfected sample was set to 1 at each dose. The graph presents the average values from triplicate experiments. Error bars represent SDs. *, P

    Article Snippet: To inhibit N-linked glycosylation, A549 cells were incubated with tunicamycin (Calbiochem) in a dose-dependent manner (0, 25, 50, and 100 μg), or lysate from A549 cells was incubated with PNGase F (New England BioLabs [NEB]).

    Techniques: Infection, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing

    Recombinant SPOCK-2 protein inhibits virus infection in vitro and in vivo . (A) Schema of the experimental procedure. (B to D) A549 cells were infected with PR8 IAV (MOI, 0.1) in the presence of medium from SPOCK2-overexpressing cells. After 12 h, the cells under the indicated conditions were collected and HA vRNA expression was detected by qRT-PCR. Each bar indicates the average value ± SD obtained from triplicate experiments. *, P

    Journal: Journal of Virology

    Article Title: The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells

    doi: 10.1128/JVI.00662-19

    Figure Lengend Snippet: Recombinant SPOCK-2 protein inhibits virus infection in vitro and in vivo . (A) Schema of the experimental procedure. (B to D) A549 cells were infected with PR8 IAV (MOI, 0.1) in the presence of medium from SPOCK2-overexpressing cells. After 12 h, the cells under the indicated conditions were collected and HA vRNA expression was detected by qRT-PCR. Each bar indicates the average value ± SD obtained from triplicate experiments. *, P

    Article Snippet: To inhibit N-linked glycosylation, A549 cells were incubated with tunicamycin (Calbiochem) in a dose-dependent manner (0, 25, 50, and 100 μg), or lysate from A549 cells was incubated with PNGase F (New England BioLabs [NEB]).

    Techniques: Recombinant, Infection, In Vitro, In Vivo, Expressing, Quantitative RT-PCR

    Influenza virus preferentially binds the sialic acid moiety of SPOCK2. (A and B) A549 cells treated with tunicamycin for 12 h (A) or PNGase F (250 U) (B) were subjected to immunoblot analysis with anti-SPOCK2, and anti-GAPDH was detected as a loading control. (C) Lysates of empty vector- or NA-expressing A549 cells infected with PR8 IAV for 12 h were subjected to immunoblot analysis with anti-SPOCK2 and anti-NA. Anti-GAPDH was detected as a loading control. (D) Purified SPOCK2 (1 μg) was incubated with PBS or PR8 IAV for 1 h and subjected to immunoblot analysis with anti-His and anti-M1. (E and F) Purified SPOCK2 (1 μg) from SPOCK2-overexpressing cells (E) or NA-co-overexpressing cells (F) analyzed by Western blotting and SNA blotting. (G) An HI assay was performed using turkey RBCs. The RBCs were added to a 96-well v-bottom plate. PR8 IAV was added with serially diluted purified SPOCK2 from the medium of SPOCK2-overexpressing cells or NA-co-overexpressing cells and incubated for 1 h at room temperature. The inhibition of PR8 IAV attachment to RBCs was then measured by observing each well. (H) Schematic illustration of the proposed model.

    Journal: Journal of Virology

    Article Title: The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells

    doi: 10.1128/JVI.00662-19

    Figure Lengend Snippet: Influenza virus preferentially binds the sialic acid moiety of SPOCK2. (A and B) A549 cells treated with tunicamycin for 12 h (A) or PNGase F (250 U) (B) were subjected to immunoblot analysis with anti-SPOCK2, and anti-GAPDH was detected as a loading control. (C) Lysates of empty vector- or NA-expressing A549 cells infected with PR8 IAV for 12 h were subjected to immunoblot analysis with anti-SPOCK2 and anti-NA. Anti-GAPDH was detected as a loading control. (D) Purified SPOCK2 (1 μg) was incubated with PBS or PR8 IAV for 1 h and subjected to immunoblot analysis with anti-His and anti-M1. (E and F) Purified SPOCK2 (1 μg) from SPOCK2-overexpressing cells (E) or NA-co-overexpressing cells (F) analyzed by Western blotting and SNA blotting. (G) An HI assay was performed using turkey RBCs. The RBCs were added to a 96-well v-bottom plate. PR8 IAV was added with serially diluted purified SPOCK2 from the medium of SPOCK2-overexpressing cells or NA-co-overexpressing cells and incubated for 1 h at room temperature. The inhibition of PR8 IAV attachment to RBCs was then measured by observing each well. (H) Schematic illustration of the proposed model.

    Article Snippet: To inhibit N-linked glycosylation, A549 cells were incubated with tunicamycin (Calbiochem) in a dose-dependent manner (0, 25, 50, and 100 μg), or lysate from A549 cells was incubated with PNGase F (New England BioLabs [NEB]).

    Techniques: Plasmid Preparation, Expressing, Infection, Purification, Incubation, Western Blot, HI Assay, Inhibition

    The heparan sulfate moiety is responsible for the antiviral effect of SPOCK2. (A) Domain deletion mutants of SPOCK2 were overexpressed in A549 cells and infected with PR8 IAV for 24 h. HA vRNA levels were measured by qRT-PCR. (B) Site-directed mutants of SPOCK2 were overexpressed in A549 cells and infected with PR8 IAV for 24 h. HA vRNA levels were measured by qRT-PCR. For the bottom portion, cell lysates from the top portion were analyzed with anti-M1 and anti-V5, and anti-GAPDH was detected as a loading control. (C) At 36 h posttransfection, A549 cells were infected with IAV at an MOI of 1 per well for 24 h or 48 h. The supernatants from cells infected with A549 cells were collected, and the viral titers were measured by the TCID 50 method. The bars indicate the mean values ± SDs obtained from three experiments. (D) A549 cells were transfected with EXT1, -2, and -3 siRNAs and a V5-tagged SPOCK2-expressing construct or empty vector. HA vRNA levels were measured by qRT-PCR. The expression was normalized by GAPDH (upper graph). The silencing efficiencies of EXT1, EXT2, and EXTL3 were measured by qRT-PCR (lower graph). Expression was normalized by GAPDH. All graphs indicate the average values ± SDs obtained from triplicate experiments. ***, P

    Journal: Journal of Virology

    Article Title: The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells

    doi: 10.1128/JVI.00662-19

    Figure Lengend Snippet: The heparan sulfate moiety is responsible for the antiviral effect of SPOCK2. (A) Domain deletion mutants of SPOCK2 were overexpressed in A549 cells and infected with PR8 IAV for 24 h. HA vRNA levels were measured by qRT-PCR. (B) Site-directed mutants of SPOCK2 were overexpressed in A549 cells and infected with PR8 IAV for 24 h. HA vRNA levels were measured by qRT-PCR. For the bottom portion, cell lysates from the top portion were analyzed with anti-M1 and anti-V5, and anti-GAPDH was detected as a loading control. (C) At 36 h posttransfection, A549 cells were infected with IAV at an MOI of 1 per well for 24 h or 48 h. The supernatants from cells infected with A549 cells were collected, and the viral titers were measured by the TCID 50 method. The bars indicate the mean values ± SDs obtained from three experiments. (D) A549 cells were transfected with EXT1, -2, and -3 siRNAs and a V5-tagged SPOCK2-expressing construct or empty vector. HA vRNA levels were measured by qRT-PCR. The expression was normalized by GAPDH (upper graph). The silencing efficiencies of EXT1, EXT2, and EXTL3 were measured by qRT-PCR (lower graph). Expression was normalized by GAPDH. All graphs indicate the average values ± SDs obtained from triplicate experiments. ***, P

    Article Snippet: To inhibit N-linked glycosylation, A549 cells were incubated with tunicamycin (Calbiochem) in a dose-dependent manner (0, 25, 50, and 100 μg), or lysate from A549 cells was incubated with PNGase F (New England BioLabs [NEB]).

    Techniques: Infection, Quantitative RT-PCR, Transfection, Expressing, Construct, Plasmid Preparation

    Purified SPOCK2 inhibits attachment and entry of influenza virus. (A) A549 cells were infected with DiI-labeled PR8 IAV with buffer or 20 μg of purified SPOCK2 for 20 min. DiI and nucleus (Hoechst) staining were examined by confocal microscopy. Representative images are shown. Scale bars, 200 μm. DIC, differential interference contrast. (B) A549 cells were infected with PR8 IAV in the presence of purified SPOCK2 (20 μg) for 20 min and then processed for subcellular fractionation. The fractions from different compartments were used for RNA isolation and the determination of HA vRNA levels by qRT-PCR. Each bar indicates the average value ± SD obtained from triplicate experiments. P values were calculated by two‐way ANOVA. *, P

    Journal: Journal of Virology

    Article Title: The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells

    doi: 10.1128/JVI.00662-19

    Figure Lengend Snippet: Purified SPOCK2 inhibits attachment and entry of influenza virus. (A) A549 cells were infected with DiI-labeled PR8 IAV with buffer or 20 μg of purified SPOCK2 for 20 min. DiI and nucleus (Hoechst) staining were examined by confocal microscopy. Representative images are shown. Scale bars, 200 μm. DIC, differential interference contrast. (B) A549 cells were infected with PR8 IAV in the presence of purified SPOCK2 (20 μg) for 20 min and then processed for subcellular fractionation. The fractions from different compartments were used for RNA isolation and the determination of HA vRNA levels by qRT-PCR. Each bar indicates the average value ± SD obtained from triplicate experiments. P values were calculated by two‐way ANOVA. *, P

    Article Snippet: To inhibit N-linked glycosylation, A549 cells were incubated with tunicamycin (Calbiochem) in a dose-dependent manner (0, 25, 50, and 100 μg), or lysate from A549 cells was incubated with PNGase F (New England BioLabs [NEB]).

    Techniques: Purification, Infection, Labeling, Staining, Confocal Microscopy, Fractionation, Isolation, Quantitative RT-PCR

    The functional significance of autophagy in A549 cells treated with fisetin (FIS) and/or paclitaxel (PTX). The A549 cells were pre-treated with bafilomycin A1 (Baf A1; 100 nM) for 4 h, followed by washing with PBS and the subsequent incubation in the absence or presence of 10 μM FIS and/or 0.1 μM PTX for 24 h. a Cell viability was determined by MTT colorimetric assay. Data are expressed as a percentage of the control. b The cytometric analysis of apoptosis using Annexin V/PI assay. The sum of the early and late apoptotic cells represented the total apoptosis. Asterisks represent statistically significant differences from control cells (p

    Journal: Cancer Cell International

    Article Title: Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells

    doi: 10.1186/s12935-016-0288-3

    Figure Lengend Snippet: The functional significance of autophagy in A549 cells treated with fisetin (FIS) and/or paclitaxel (PTX). The A549 cells were pre-treated with bafilomycin A1 (Baf A1; 100 nM) for 4 h, followed by washing with PBS and the subsequent incubation in the absence or presence of 10 μM FIS and/or 0.1 μM PTX for 24 h. a Cell viability was determined by MTT colorimetric assay. Data are expressed as a percentage of the control. b The cytometric analysis of apoptosis using Annexin V/PI assay. The sum of the early and late apoptotic cells represented the total apoptosis. Asterisks represent statistically significant differences from control cells (p

    Article Snippet: Detection and quantification of acidic vesicular organelles with acridine orange staining To detect the development of acidic vesicular organelles (AVOs), which are the hallmark of autophagy, the vital staining of A549 cells with acridine orange (AO; Sigma-Aldrich; St. Louis, MO, USA) was performed.

    Techniques: Functional Assay, Incubation, MTT Assay, Colorimetric Assay

    The detection of autophagy by LC3 accumulation. a – o Fluorescent microscope was used to visualize the LC3-II punctate staining pattern ( green ); the cell nuclei were counterstained with DAPI ( blue ). a – l The A549 cells were treated with 10 µM fisetin (FIS) and/or 0.1 µM paclitaxel (PTX) for 24 h or left untreated (control, CTRL). m – o As negative control, bafilomycin A1 (Baf A1; 100 nM) was added to the cells for a period of 4 h, followed by washing with PBS and subsequent incubation with FIS and PTX for 24 h. Note the increase in LC3-II puncta following FIS and PTX treatment in the absence or presence of Baf A1 ( j , m ). Bar 50 µm. p Real-time PCR measurement of LC3-II mRNA expression in A549 cells. The expression was normalized to glucose 6-phosphate dehydrogenase (G6PD) and presented as a fold difference relative to a calibrator sample (untreated A549 cells; designated as 1). Error bars represent standard deviation from duplicate qRT-PCR assays

    Journal: Cancer Cell International

    Article Title: Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells

    doi: 10.1186/s12935-016-0288-3

    Figure Lengend Snippet: The detection of autophagy by LC3 accumulation. a – o Fluorescent microscope was used to visualize the LC3-II punctate staining pattern ( green ); the cell nuclei were counterstained with DAPI ( blue ). a – l The A549 cells were treated with 10 µM fisetin (FIS) and/or 0.1 µM paclitaxel (PTX) for 24 h or left untreated (control, CTRL). m – o As negative control, bafilomycin A1 (Baf A1; 100 nM) was added to the cells for a period of 4 h, followed by washing with PBS and subsequent incubation with FIS and PTX for 24 h. Note the increase in LC3-II puncta following FIS and PTX treatment in the absence or presence of Baf A1 ( j , m ). Bar 50 µm. p Real-time PCR measurement of LC3-II mRNA expression in A549 cells. The expression was normalized to glucose 6-phosphate dehydrogenase (G6PD) and presented as a fold difference relative to a calibrator sample (untreated A549 cells; designated as 1). Error bars represent standard deviation from duplicate qRT-PCR assays

    Article Snippet: Detection and quantification of acidic vesicular organelles with acridine orange staining To detect the development of acidic vesicular organelles (AVOs), which are the hallmark of autophagy, the vital staining of A549 cells with acridine orange (AO; Sigma-Aldrich; St. Louis, MO, USA) was performed.

    Techniques: Microscopy, Staining, Negative Control, Incubation, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, Quantitative RT-PCR

    The detection of autophagy by acridine orange staining (AO). a – n Fluorescent microscope was used to visualize the acidic vesicular organelles (AVOs; red fluorescence ) as well as the cytoplasm and nucleus ( green fluorescence ) after the vital staining of the cells with AO, as indicated in Materials and methods. a – h The A549 cells were treated with 10 µM fisetin (FIS) and/or 0.1 µM paclitaxel (PTX) for 24 h or left untreated (control, CTRL). i , j As negative control, bafilomycin A1 (Baf A1; 100 nM) was added to the cells for a period of 4 h, followed by washing with PBS and subsequent incubation with FIS and PTX for 24 h. k – n In another set of experiment, the cells were treated with the combination of 10 µM fisetin and 0.1 µM paclitaxel for 24 h, followed by post-treatment incubation in a drug-free medium for the next 24 or 48 h. Note the increased amount of AVOs after the treatment with FIS and/or PTX ( c , e , g ). Arrowheads indicate: ( I ) the giant multinucleated cells filled with numerous AVOs. Bar 50 µm. o The measurement of the red fluorescence of AO using image-based cytometer. Asterisks represent statistically significant differences from control cells, and symbol ^ indicates statistically significant differences compared to the treatment with FIS plus PTX (p

    Journal: Cancer Cell International

    Article Title: Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells

    doi: 10.1186/s12935-016-0288-3

    Figure Lengend Snippet: The detection of autophagy by acridine orange staining (AO). a – n Fluorescent microscope was used to visualize the acidic vesicular organelles (AVOs; red fluorescence ) as well as the cytoplasm and nucleus ( green fluorescence ) after the vital staining of the cells with AO, as indicated in Materials and methods. a – h The A549 cells were treated with 10 µM fisetin (FIS) and/or 0.1 µM paclitaxel (PTX) for 24 h or left untreated (control, CTRL). i , j As negative control, bafilomycin A1 (Baf A1; 100 nM) was added to the cells for a period of 4 h, followed by washing with PBS and subsequent incubation with FIS and PTX for 24 h. k – n In another set of experiment, the cells were treated with the combination of 10 µM fisetin and 0.1 µM paclitaxel for 24 h, followed by post-treatment incubation in a drug-free medium for the next 24 or 48 h. Note the increased amount of AVOs after the treatment with FIS and/or PTX ( c , e , g ). Arrowheads indicate: ( I ) the giant multinucleated cells filled with numerous AVOs. Bar 50 µm. o The measurement of the red fluorescence of AO using image-based cytometer. Asterisks represent statistically significant differences from control cells, and symbol ^ indicates statistically significant differences compared to the treatment with FIS plus PTX (p

    Article Snippet: Detection and quantification of acidic vesicular organelles with acridine orange staining To detect the development of acidic vesicular organelles (AVOs), which are the hallmark of autophagy, the vital staining of A549 cells with acridine orange (AO; Sigma-Aldrich; St. Louis, MO, USA) was performed.

    Techniques: Staining, Microscopy, Fluorescence, Negative Control, Incubation, Cytometry

    Mitosis and cytokinesis abnormalities in A549 cells after treatment with fisetin (FIS) and/or paclitaxel (PTX). The A549 cells were treated with 10 µM FIS and/or 0.1 µM PTX for 24 h or left untreated (CTRL). a – l Mitotic spindles were stained with β-tubulin antibody in red color and DNA was stained by DAPI in blue color and visualized by fluorescence microscope. Arrowheads indicate: normal mitotic figures in control ( a – c ) and FIS-treated ( d – f ) cells ( I ) bipolar spindles; ( II ) metaphase cells with chromosomes aligned on the metaphase plate; ( III ) anaphase cells; ( IV ) late telophase/cytokinesis; mitosis and cytokinesis defects in PTX-treated ( g – i ) and co-treated ( j – l ) cells ( V ) monopolar spindles; ( VI ) multipolar spindles; ( VII ) abnormal chromosome alignments and segregation; ( VIII ) multipolar cell division resulting from multipolar spindles; ( IX ) cleavage failure as a consequence of multipolar spindles; ( X ) multinucleated or mononucleated interphase cells as a morphological manifestation of mitotic slippage. Bar 50 µm. m The quantification of spindles abnormalities. The percentage of cells containing normal bipolar spindles ( light grey columns ), multipolar spindles ( dark grey columns ), and monopolar spindles ( white columns ) was evaluated. At least 100 cells were counted on each of the three slides. Asterisks represent statistically significant differences from control cells (p

    Journal: Cancer Cell International

    Article Title: Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells

    doi: 10.1186/s12935-016-0288-3

    Figure Lengend Snippet: Mitosis and cytokinesis abnormalities in A549 cells after treatment with fisetin (FIS) and/or paclitaxel (PTX). The A549 cells were treated with 10 µM FIS and/or 0.1 µM PTX for 24 h or left untreated (CTRL). a – l Mitotic spindles were stained with β-tubulin antibody in red color and DNA was stained by DAPI in blue color and visualized by fluorescence microscope. Arrowheads indicate: normal mitotic figures in control ( a – c ) and FIS-treated ( d – f ) cells ( I ) bipolar spindles; ( II ) metaphase cells with chromosomes aligned on the metaphase plate; ( III ) anaphase cells; ( IV ) late telophase/cytokinesis; mitosis and cytokinesis defects in PTX-treated ( g – i ) and co-treated ( j – l ) cells ( V ) monopolar spindles; ( VI ) multipolar spindles; ( VII ) abnormal chromosome alignments and segregation; ( VIII ) multipolar cell division resulting from multipolar spindles; ( IX ) cleavage failure as a consequence of multipolar spindles; ( X ) multinucleated or mononucleated interphase cells as a morphological manifestation of mitotic slippage. Bar 50 µm. m The quantification of spindles abnormalities. The percentage of cells containing normal bipolar spindles ( light grey columns ), multipolar spindles ( dark grey columns ), and monopolar spindles ( white columns ) was evaluated. At least 100 cells were counted on each of the three slides. Asterisks represent statistically significant differences from control cells (p

    Article Snippet: Detection and quantification of acidic vesicular organelles with acridine orange staining To detect the development of acidic vesicular organelles (AVOs), which are the hallmark of autophagy, the vital staining of A549 cells with acridine orange (AO; Sigma-Aldrich; St. Louis, MO, USA) was performed.

    Techniques: Staining, Fluorescence, Microscopy

    The detection of autophagy using transmission electron microscope. The A549 cells were treated with 10 µM fisetin (FIS) and/or 0.1 µM paclitaxel (PTX) for 24 h or left untreated (control, CTRL). Arrowheads indicate: ( I ) empty autophagic-like vacuoles; ( II ) autophagic-like vacuoles filled with the amorphous materials, the membranous inclusions or the organelles at the various stages of degradation; ( III ) single-membrane vacuoles (autolysosome-like structures); ( IV ) the swollen mitochondria with distorted or disorganized cristae; ( V ) large lysosome-like structure with electron-dense material. Representative micrographs of two independent experiments. Bar 2 µm

    Journal: Cancer Cell International

    Article Title: Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells

    doi: 10.1186/s12935-016-0288-3

    Figure Lengend Snippet: The detection of autophagy using transmission electron microscope. The A549 cells were treated with 10 µM fisetin (FIS) and/or 0.1 µM paclitaxel (PTX) for 24 h or left untreated (control, CTRL). Arrowheads indicate: ( I ) empty autophagic-like vacuoles; ( II ) autophagic-like vacuoles filled with the amorphous materials, the membranous inclusions or the organelles at the various stages of degradation; ( III ) single-membrane vacuoles (autolysosome-like structures); ( IV ) the swollen mitochondria with distorted or disorganized cristae; ( V ) large lysosome-like structure with electron-dense material. Representative micrographs of two independent experiments. Bar 2 µm

    Article Snippet: Detection and quantification of acidic vesicular organelles with acridine orange staining To detect the development of acidic vesicular organelles (AVOs), which are the hallmark of autophagy, the vital staining of A549 cells with acridine orange (AO; Sigma-Aldrich; St. Louis, MO, USA) was performed.

    Techniques: Transmission Assay, Microscopy

    The combined effect of fisetin (FIS) and paclitaxel (PTX) on the A549 cells. a The cells were treated with various concentrations of PTX (0.1, 0.2, 0.3, 0.4, 0.5 μM) and FIS (10, 20, 30, 40, 50 µM), either alone or in a fixed ratio of 1:100, for 24 h. Cell viability was determined by MTT colorimetric assay. Data are expressed as a percentage of the control. Symbols $ and # indicate statistically significant differences compared with FIS or PTX treatment alone, respectively (p

    Journal: Cancer Cell International

    Article Title: Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells

    doi: 10.1186/s12935-016-0288-3

    Figure Lengend Snippet: The combined effect of fisetin (FIS) and paclitaxel (PTX) on the A549 cells. a The cells were treated with various concentrations of PTX (0.1, 0.2, 0.3, 0.4, 0.5 μM) and FIS (10, 20, 30, 40, 50 µM), either alone or in a fixed ratio of 1:100, for 24 h. Cell viability was determined by MTT colorimetric assay. Data are expressed as a percentage of the control. Symbols $ and # indicate statistically significant differences compared with FIS or PTX treatment alone, respectively (p

    Article Snippet: Detection and quantification of acidic vesicular organelles with acridine orange staining To detect the development of acidic vesicular organelles (AVOs), which are the hallmark of autophagy, the vital staining of A549 cells with acridine orange (AO; Sigma-Aldrich; St. Louis, MO, USA) was performed.

    Techniques: MTT Assay, Colorimetric Assay

    The assessment of the nuclear morphology using DAPI staining and fluorescence microscopy. The A549 cells were incubated with 10 µM fisetin (FIS), 0.1 µM paclitaxel (PTX) either as single agents or in the combination for 6, 10 or 30 h. Micrographs are representative of three independent experiments. Arrowheads indicate: ( I ) the shrunken or fragmented nuclei with chromatin condensation; the enlarged multinucleated ( II ) or mononucleated ( III ) cells. Bar 50 µm

    Journal: Cancer Cell International

    Article Title: Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells

    doi: 10.1186/s12935-016-0288-3

    Figure Lengend Snippet: The assessment of the nuclear morphology using DAPI staining and fluorescence microscopy. The A549 cells were incubated with 10 µM fisetin (FIS), 0.1 µM paclitaxel (PTX) either as single agents or in the combination for 6, 10 or 30 h. Micrographs are representative of three independent experiments. Arrowheads indicate: ( I ) the shrunken or fragmented nuclei with chromatin condensation; the enlarged multinucleated ( II ) or mononucleated ( III ) cells. Bar 50 µm

    Article Snippet: Detection and quantification of acidic vesicular organelles with acridine orange staining To detect the development of acidic vesicular organelles (AVOs), which are the hallmark of autophagy, the vital staining of A549 cells with acridine orange (AO; Sigma-Aldrich; St. Louis, MO, USA) was performed.

    Techniques: Staining, Fluorescence, Microscopy, Incubation

    The effect of fisetin (FIS) and/or paclitaxel (PTX) on apoptosis of A549 cells. a , b Annexin V/PI assay by image-based cytometry. The sum of the early and late apoptotic cells represented the total apoptosis. a , c , d , e The A549 cells were treated with 10 µM FIS and/or 0.1 µM PTX for 24 h. b In another set of experiment, the cells were treated with the combination of 10 µM fisetin and 0.1 µM paclitaxel for 30 h, followed by post-treatment incubation in a drug-free medium for the next 24 or 48 h. Real-time PCR measurement of c caspase-3, d Bax, e Bcl-2 mRNA expression in A549 cells. The expression was normalized to GAPDH and presented as a fold difference relative to a calibrator sample (untreated A549 cells; designated as 1). f Bcl-2/Bax ratio. Control cells (CTRL) were cultured under identical conditions, but without the addition of the tested agents. Asterisks represent statistically significant differences from control cells (p

    Journal: Cancer Cell International

    Article Title: Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells

    doi: 10.1186/s12935-016-0288-3

    Figure Lengend Snippet: The effect of fisetin (FIS) and/or paclitaxel (PTX) on apoptosis of A549 cells. a , b Annexin V/PI assay by image-based cytometry. The sum of the early and late apoptotic cells represented the total apoptosis. a , c , d , e The A549 cells were treated with 10 µM FIS and/or 0.1 µM PTX for 24 h. b In another set of experiment, the cells were treated with the combination of 10 µM fisetin and 0.1 µM paclitaxel for 30 h, followed by post-treatment incubation in a drug-free medium for the next 24 or 48 h. Real-time PCR measurement of c caspase-3, d Bax, e Bcl-2 mRNA expression in A549 cells. The expression was normalized to GAPDH and presented as a fold difference relative to a calibrator sample (untreated A549 cells; designated as 1). f Bcl-2/Bax ratio. Control cells (CTRL) were cultured under identical conditions, but without the addition of the tested agents. Asterisks represent statistically significant differences from control cells (p

    Article Snippet: Detection and quantification of acidic vesicular organelles with acridine orange staining To detect the development of acidic vesicular organelles (AVOs), which are the hallmark of autophagy, the vital staining of A549 cells with acridine orange (AO; Sigma-Aldrich; St. Louis, MO, USA) was performed.

    Techniques: Cytometry, Incubation, Real-time Polymerase Chain Reaction, Expressing, Cell Culture

    Miltirone induces apoptosis in HCC827 and A549 cisplatin-resistant lung cancer cells. **P

    Journal: Oncology Letters

    Article Title: Miltirone-induced apoptosis in cisplatin-resistant lung cancer cells through upregulation of p53 signaling pathways

    doi: 10.3892/ol.2018.8440

    Figure Lengend Snippet: Miltirone induces apoptosis in HCC827 and A549 cisplatin-resistant lung cancer cells. **P

    Article Snippet: HCC827 and A549 cells were lysed with a radioimmunoprecipitation assay (Sigma-Aldrich; Merck KGaA) for 30 min on ice.

    Techniques:

    Miltirone decreases the viability of (A) HCC827 and (B) A549 cisplatin-resistant lung cancer cells. **P

    Journal: Oncology Letters

    Article Title: Miltirone-induced apoptosis in cisplatin-resistant lung cancer cells through upregulation of p53 signaling pathways

    doi: 10.3892/ol.2018.8440

    Figure Lengend Snippet: Miltirone decreases the viability of (A) HCC827 and (B) A549 cisplatin-resistant lung cancer cells. **P

    Article Snippet: HCC827 and A549 cells were lysed with a radioimmunoprecipitation assay (Sigma-Aldrich; Merck KGaA) for 30 min on ice.

    Techniques:

    Miltirone decreases mitochondrial ROS generation in HCC827 and A549 cisplatin-resistant lung cancer cells. **P

    Journal: Oncology Letters

    Article Title: Miltirone-induced apoptosis in cisplatin-resistant lung cancer cells through upregulation of p53 signaling pathways

    doi: 10.3892/ol.2018.8440

    Figure Lengend Snippet: Miltirone decreases mitochondrial ROS generation in HCC827 and A549 cisplatin-resistant lung cancer cells. **P

    Article Snippet: HCC827 and A549 cells were lysed with a radioimmunoprecipitation assay (Sigma-Aldrich; Merck KGaA) for 30 min on ice.

    Techniques:

    Miltirone induces Bax, AIF and p53 protein expression in HCC827 and A549 cisplatin-resistant lung cancer cells. (A) The effect of miltirone on Bax, AIF and p53 protein expression was determined using western blotting in HCC827 and A549 cisplatin-resistant lung cancer cells. The statistical analysis of (B) Bax, (C) AIF and (D) p53 protein expression. GAPDH served as a loading control. **P

    Journal: Oncology Letters

    Article Title: Miltirone-induced apoptosis in cisplatin-resistant lung cancer cells through upregulation of p53 signaling pathways

    doi: 10.3892/ol.2018.8440

    Figure Lengend Snippet: Miltirone induces Bax, AIF and p53 protein expression in HCC827 and A549 cisplatin-resistant lung cancer cells. (A) The effect of miltirone on Bax, AIF and p53 protein expression was determined using western blotting in HCC827 and A549 cisplatin-resistant lung cancer cells. The statistical analysis of (B) Bax, (C) AIF and (D) p53 protein expression. GAPDH served as a loading control. **P

    Article Snippet: HCC827 and A549 cells were lysed with a radioimmunoprecipitation assay (Sigma-Aldrich; Merck KGaA) for 30 min on ice.

    Techniques: Expressing, Western Blot

    Miltirone decreases MMP2/9 and increases PARP protein expression in HCC827 and A549 cisplatin-resistant lung cancer cells. (A) The effect of miltirone on MMP2/9 and PARP protein expression was determined using western blotting in HCC827 and A549 cisplatin-resistant lung cancer cells. The statistical analysis of (B) MMP2, (C) MMP9 and (D)PARP protein expression. GAPDH served as a loading control. **P

    Journal: Oncology Letters

    Article Title: Miltirone-induced apoptosis in cisplatin-resistant lung cancer cells through upregulation of p53 signaling pathways

    doi: 10.3892/ol.2018.8440

    Figure Lengend Snippet: Miltirone decreases MMP2/9 and increases PARP protein expression in HCC827 and A549 cisplatin-resistant lung cancer cells. (A) The effect of miltirone on MMP2/9 and PARP protein expression was determined using western blotting in HCC827 and A549 cisplatin-resistant lung cancer cells. The statistical analysis of (B) MMP2, (C) MMP9 and (D)PARP protein expression. GAPDH served as a loading control. **P

    Article Snippet: HCC827 and A549 cells were lysed with a radioimmunoprecipitation assay (Sigma-Aldrich; Merck KGaA) for 30 min on ice.

    Techniques: Expressing, Western Blot

    Miltirone decreases caspase-3/8 activity in (A) HCC827 and (B) A549 cisplatin-resistant lung cancer cells. **P

    Journal: Oncology Letters

    Article Title: Miltirone-induced apoptosis in cisplatin-resistant lung cancer cells through upregulation of p53 signaling pathways

    doi: 10.3892/ol.2018.8440

    Figure Lengend Snippet: Miltirone decreases caspase-3/8 activity in (A) HCC827 and (B) A549 cisplatin-resistant lung cancer cells. **P

    Article Snippet: HCC827 and A549 cells were lysed with a radioimmunoprecipitation assay (Sigma-Aldrich; Merck KGaA) for 30 min on ice.

    Techniques: Activity Assay

    Lentivirus-delivered FGFR1 overexpression affected influenza A virus internalization, but not binding, during virus entry. A549 cells with FGFR1, FGFR4, or GFP overexpression were pretreated with or without sialidase, and then infected with influenza A/PR8 ( A ) or H5N1 ( B ). The procedures were described in detail in the Methods, virus binding and internalization assay. Influenza virus NP was detected by Western blotting using anti-influenza NP antibodies. β-actin protein was used as an internal control. The ratio of NP/β-actin was determined based on densitometric analysis. The results were expressed as means ± s.e.m. (n = 3). Values of P

    Journal: PLoS ONE

    Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

    doi: 10.1371/journal.pone.0124651

    Figure Lengend Snippet: Lentivirus-delivered FGFR1 overexpression affected influenza A virus internalization, but not binding, during virus entry. A549 cells with FGFR1, FGFR4, or GFP overexpression were pretreated with or without sialidase, and then infected with influenza A/PR8 ( A ) or H5N1 ( B ). The procedures were described in detail in the Methods, virus binding and internalization assay. Influenza virus NP was detected by Western blotting using anti-influenza NP antibodies. β-actin protein was used as an internal control. The ratio of NP/β-actin was determined based on densitometric analysis. The results were expressed as means ± s.e.m. (n = 3). Values of P

    Article Snippet: For lentivirus infection, A549 cells were incubated with diluted virus supernatant ( > 95% infection efficiency) supplemented with 6 μg polybrene (Sigma-Aldrich) ml-1 for 8 h. Medium was then replaced with fresh complete medium and cultured for a further 40 h. Cells were harvested for FGFR ectopic expression efficiency determination by real-time PCR and Western blotting assay.

    Techniques: Over Expression, Binding Assay, Infection, Western Blot

    Time-course analysis of FGFR family member expression in A549 cells infected with influenza A/PR/8/34 virus. ( A - D ) A549 cells were infected with PR8 virus at an MOI of 1 for the indicated times. FGFR mRNA levels were detected using real-time PCR analysis. The mRNA expression of FGFR1 ( A ), FGFR2 ( B ), FGFR3 ( C ), and FGFR4 ( D ) relative to the reference gene GAPDH was calculated. ( E , F ) The lysates of A549 cells were obtained at the indicated times post-PR8 infection. Protein levels of FGFR1 ( E ) and FGFR4 ( F ) were determined by Western blotting using specific antibodies. Densitometric analysis relative to β-actin levels was expressed as fold change. All graphs present the means ± s.e.m. (n = 3). Values of P

    Journal: PLoS ONE

    Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

    doi: 10.1371/journal.pone.0124651

    Figure Lengend Snippet: Time-course analysis of FGFR family member expression in A549 cells infected with influenza A/PR/8/34 virus. ( A - D ) A549 cells were infected with PR8 virus at an MOI of 1 for the indicated times. FGFR mRNA levels were detected using real-time PCR analysis. The mRNA expression of FGFR1 ( A ), FGFR2 ( B ), FGFR3 ( C ), and FGFR4 ( D ) relative to the reference gene GAPDH was calculated. ( E , F ) The lysates of A549 cells were obtained at the indicated times post-PR8 infection. Protein levels of FGFR1 ( E ) and FGFR4 ( F ) were determined by Western blotting using specific antibodies. Densitometric analysis relative to β-actin levels was expressed as fold change. All graphs present the means ± s.e.m. (n = 3). Values of P

    Article Snippet: For lentivirus infection, A549 cells were incubated with diluted virus supernatant ( > 95% infection efficiency) supplemented with 6 μg polybrene (Sigma-Aldrich) ml-1 for 8 h. Medium was then replaced with fresh complete medium and cultured for a further 40 h. Cells were harvested for FGFR ectopic expression efficiency determination by real-time PCR and Western blotting assay.

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Western Blot

    Lentivirus-mediated FGFR1 overexpression in A549 cells significantly decreased influenza A virus replication. A549 cells were infected with recombinant lentivirus expressing FGFR1, FGFR4, or GFP (as a control). After 48 h, A549 cells were infected with PR8 virus (MOI = 1). The supernatants and lysates of A549 cells were harvested after 24 h virus infection. ( A , B ) Influenza M1 mRNA levels in A549 cells with PR8 and H5N1 were detected using real-time PCR. ( C , D ) Progeny virus titers of PR8 and H5N1 were determined as described previously. ( E , F ) FGFR1 and FGFR4 expression efficiencies were detected using real-time PCR and Western blotting. All graphs present the means ± s.e.m. (n = 3). Values of P

    Journal: PLoS ONE

    Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

    doi: 10.1371/journal.pone.0124651

    Figure Lengend Snippet: Lentivirus-mediated FGFR1 overexpression in A549 cells significantly decreased influenza A virus replication. A549 cells were infected with recombinant lentivirus expressing FGFR1, FGFR4, or GFP (as a control). After 48 h, A549 cells were infected with PR8 virus (MOI = 1). The supernatants and lysates of A549 cells were harvested after 24 h virus infection. ( A , B ) Influenza M1 mRNA levels in A549 cells with PR8 and H5N1 were detected using real-time PCR. ( C , D ) Progeny virus titers of PR8 and H5N1 were determined as described previously. ( E , F ) FGFR1 and FGFR4 expression efficiencies were detected using real-time PCR and Western blotting. All graphs present the means ± s.e.m. (n = 3). Values of P

    Article Snippet: For lentivirus infection, A549 cells were incubated with diluted virus supernatant ( > 95% infection efficiency) supplemented with 6 μg polybrene (Sigma-Aldrich) ml-1 for 8 h. Medium was then replaced with fresh complete medium and cultured for a further 40 h. Cells were harvested for FGFR ectopic expression efficiency determination by real-time PCR and Western blotting assay.

    Techniques: Over Expression, Infection, Recombinant, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Repression of FGFR1 phosphorylation by the PD173074 inhibitor enhanced influenza A/PR8virus replication. (A) The lysates of A549 cells with PR8 virus infection were harvested for the indicated times. FGFR1 phosphorylation levels were determined by Western blotting with phospho-specific antibodies. Densitometric analysis of the phosphorylated/total FGFR1 ratio was shown as fold change. (B-D) A549 cells were pretreated with PD173074 (FGFR1 kinase inhibitor) at indicated concentrations for 30 min, and then infected with PR8 (indicated-dose inhibitor was also added to virus diluent). After 1 h, the medium was replaced with fresh medium supplemented with PD173074. (B) The cell lysates were collected at 12 hpi and then subjected to Western blotting for FGFR1 phosphorylation detection. The phosphorylated/total FGFR1 ratio indicated the fold change of FGFR1 phosphorylation. (C) Influenza M1 mRNA in PR8 infected-A549 cells with PD173074 treatment as indicated was detected by real-time PCR. (D) MTT assay of A549 cells with PD173074 treatment as indicated with or without PR8 infection. The results were expressed as means ± s.e.m. (n = 3). Values of P

    Journal: PLoS ONE

    Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

    doi: 10.1371/journal.pone.0124651

    Figure Lengend Snippet: Repression of FGFR1 phosphorylation by the PD173074 inhibitor enhanced influenza A/PR8virus replication. (A) The lysates of A549 cells with PR8 virus infection were harvested for the indicated times. FGFR1 phosphorylation levels were determined by Western blotting with phospho-specific antibodies. Densitometric analysis of the phosphorylated/total FGFR1 ratio was shown as fold change. (B-D) A549 cells were pretreated with PD173074 (FGFR1 kinase inhibitor) at indicated concentrations for 30 min, and then infected with PR8 (indicated-dose inhibitor was also added to virus diluent). After 1 h, the medium was replaced with fresh medium supplemented with PD173074. (B) The cell lysates were collected at 12 hpi and then subjected to Western blotting for FGFR1 phosphorylation detection. The phosphorylated/total FGFR1 ratio indicated the fold change of FGFR1 phosphorylation. (C) Influenza M1 mRNA in PR8 infected-A549 cells with PD173074 treatment as indicated was detected by real-time PCR. (D) MTT assay of A549 cells with PD173074 treatment as indicated with or without PR8 infection. The results were expressed as means ± s.e.m. (n = 3). Values of P

    Article Snippet: For lentivirus infection, A549 cells were incubated with diluted virus supernatant ( > 95% infection efficiency) supplemented with 6 μg polybrene (Sigma-Aldrich) ml-1 for 8 h. Medium was then replaced with fresh complete medium and cultured for a further 40 h. Cells were harvested for FGFR ectopic expression efficiency determination by real-time PCR and Western blotting assay.

    Techniques: Infection, Western Blot, Real-time Polymerase Chain Reaction, MTT Assay

    Specific siRNA target FGFR1 markedly increased PR8 virus entry at an early stage of the viral life cycle. ( A - C ) A549 cells were transfected with FGFR1 siRNA#1, FGFR4 siRNA#1, and negative control siRNA. After 48 h of transduction, A549 cells were incubated with PR8 virus at an MOI of 0.01 for 4 h, followed by indirect immunofluorescence assays. A549 cells were stained with anti-influenza A virus NP antibodies (green) and Hoechst 33342 (nucleus, blue). ( D ) The data of PR8-infected cells were presented as the percentages of NP-positive cells to the total number of cells. The bars represent the means ± s.e.m. (n = 3). P

    Journal: PLoS ONE

    Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

    doi: 10.1371/journal.pone.0124651

    Figure Lengend Snippet: Specific siRNA target FGFR1 markedly increased PR8 virus entry at an early stage of the viral life cycle. ( A - C ) A549 cells were transfected with FGFR1 siRNA#1, FGFR4 siRNA#1, and negative control siRNA. After 48 h of transduction, A549 cells were incubated with PR8 virus at an MOI of 0.01 for 4 h, followed by indirect immunofluorescence assays. A549 cells were stained with anti-influenza A virus NP antibodies (green) and Hoechst 33342 (nucleus, blue). ( D ) The data of PR8-infected cells were presented as the percentages of NP-positive cells to the total number of cells. The bars represent the means ± s.e.m. (n = 3). P

    Article Snippet: For lentivirus infection, A549 cells were incubated with diluted virus supernatant ( > 95% infection efficiency) supplemented with 6 μg polybrene (Sigma-Aldrich) ml-1 for 8 h. Medium was then replaced with fresh complete medium and cultured for a further 40 h. Cells were harvested for FGFR ectopic expression efficiency determination by real-time PCR and Western blotting assay.

    Techniques: Transfection, Negative Control, Transduction, Incubation, Immunofluorescence, Staining, Infection

    FGFR1 silencing by RNAi increased influenza A/PR8 and H5N1 virus replication. ( A - D ) A549 cells were transiently transfected with specific siRNA targeting FGFR1, FGFR4, or negative control siRNA. Forty-eight hours later, A549 cells were infected with PR8 virus at an MOI of 1. The cell culture supernatants and cell lysates were obtained at 24 hpi. Influenza virus M1 mRNA expression in A549 cells with PR8 ( A ) or H5N1 ( B ) infection was detected using real-time PCR. Progeny virus titers of PR8 ( C ) or H5N1 ( D ) were determined using MDCK cells with the TCID 50 assay. ( E ) The knockdown efficiencies of FGFR1 and FGFR4 by target siRNA were tested using real-time PCR. ( F ) Protein expressions of FGFR1 and FGFR4 were detected using specific antibodies by Western blotting assay. All graphs represent the means ± s.e.m. (n = 3). Values of P

    Journal: PLoS ONE

    Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

    doi: 10.1371/journal.pone.0124651

    Figure Lengend Snippet: FGFR1 silencing by RNAi increased influenza A/PR8 and H5N1 virus replication. ( A - D ) A549 cells were transiently transfected with specific siRNA targeting FGFR1, FGFR4, or negative control siRNA. Forty-eight hours later, A549 cells were infected with PR8 virus at an MOI of 1. The cell culture supernatants and cell lysates were obtained at 24 hpi. Influenza virus M1 mRNA expression in A549 cells with PR8 ( A ) or H5N1 ( B ) infection was detected using real-time PCR. Progeny virus titers of PR8 ( C ) or H5N1 ( D ) were determined using MDCK cells with the TCID 50 assay. ( E ) The knockdown efficiencies of FGFR1 and FGFR4 by target siRNA were tested using real-time PCR. ( F ) Protein expressions of FGFR1 and FGFR4 were detected using specific antibodies by Western blotting assay. All graphs represent the means ± s.e.m. (n = 3). Values of P

    Article Snippet: For lentivirus infection, A549 cells were incubated with diluted virus supernatant ( > 95% infection efficiency) supplemented with 6 μg polybrene (Sigma-Aldrich) ml-1 for 8 h. Medium was then replaced with fresh complete medium and cultured for a further 40 h. Cells were harvested for FGFR ectopic expression efficiency determination by real-time PCR and Western blotting assay.

    Techniques: Transfection, Negative Control, Infection, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot