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  • 99
    Millipore epithermal growth factor
    Npnt regulates Sox2 expression via <t>EGF</t> signaling pathway. ( A ) Ratio of Sox2+ cells among M3H1 cells transfected with Npnt-FL, with or without EGFR siRNA. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. ( B ) qRT-PCR analysis of Sox2 expression in M3H1 cells transfected with Npnt-FL, with or without EGFR siRNA. Gapdh was used as the internal control. ( C ) Ratio of Sox2 cells among M3H1 cells transfected with Npnt-FL, or treated with EGFR inhibitor <t>gefitinib</t> or lapatinib. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. ( D ) qRT-PCR analysis of Sox2 expression in M3H1 cells transfected with Npnt-FL, or treated with EGFR inhibitor gefitinib or lapatinib. Gapdh was used as the internal control. *P
    Epithermal Growth Factor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/epithermal growth factor/product/Millipore
    Average 99 stars, based on 43 article reviews
    Price from $9.99 to $1999.99
    epithermal growth factor - by Bioz Stars, 2020-05
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    85
    Upstate Biotechnology Inc growth factor epidermal growth factor egf
    Npnt regulates Sox2 expression via <t>EGF</t> signaling pathway. ( A ) Ratio of Sox2+ cells among M3H1 cells transfected with Npnt-FL, with or without EGFR siRNA. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. ( B ) qRT-PCR analysis of Sox2 expression in M3H1 cells transfected with Npnt-FL, with or without EGFR siRNA. Gapdh was used as the internal control. ( C ) Ratio of Sox2 cells among M3H1 cells transfected with Npnt-FL, or treated with EGFR inhibitor <t>gefitinib</t> or lapatinib. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. ( D ) qRT-PCR analysis of Sox2 expression in M3H1 cells transfected with Npnt-FL, or treated with EGFR inhibitor gefitinib or lapatinib. Gapdh was used as the internal control. *P
    Growth Factor Epidermal Growth Factor Egf, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/growth factor epidermal growth factor egf/product/Upstate Biotechnology Inc
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    99
    Millipore growth factor
    Causal network analysis, the identification and validation of pro-invasive growth-factors in the invasion signature of fibroblasts. A heatmap that indicates a ranking of physiological upstream regulators (filtered for ‘growth factors’) according to their p-values for gene expression data at 72 and 96 hours upon invasion ( a ). The predicted activated upstream regulators were functionally confirmed in vitro by a 3D invasion assay. Therefore, the invasive capacity of MLg fibroblasts was assessed by software-based quantification of the number of fibroblast nuclei (stained with DAPI) within and on top of the collagen matrix of 3D reconstructed confocal z-stacks. Fibroblasts were treated and left to invade the 3D collagen matrices for 48 hours. <t>TGFβ1</t> (1 and 5 ng/ml), FGF2 (10 and 50 ng/ml), PDGF-BB (5 and 25 ng/ml) and <t>EGF</t> (10 and 50 ng/ml) were tested in the invasion assay ( b - e ). The data shown represent mean values (s.d.) from three to five independent experiment (n = 3–5) including five technical replicates each. (n.t. = non-treated). Statistical analysis: One way ANOVA with Dunnett’s multiple comparison test. *p
    Growth Factor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/growth factor/product/Millipore
    Average 99 stars, based on 42 article reviews
    Price from $9.99 to $1999.99
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    99
    Millipore egf
    (A) <t>Cyclopamine</t> inhibits expression of PTCH and GLI1 RNA in LNCaP C4-2B cells: control cells (lanes 1 and 3); 24 hrs 14 nM cyclopamine treatment (lanes 2 and 4). Relative expression determined by QPCR (error bars show standard deviation). (B) Immunoblot showing inhibitory effect of 168 nM gefitinib on EGFR phosphorylation following <t>EGF</t> treatment in AIPC cells (lane 1: control; lane 2: 24 hr gefitinib; lane 3: 48 hr gefitinib). (C) Immunoblot showing inhibitory effect of 102 nM lapatinib on EGFR phosphorylation following EGF treatment in AIPC cells (lane 1: control; lane 2: 24 hr lapatinib; lane 3: 48 hr lapatinib).
    Egf, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5650 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egf/product/Millipore
    Average 99 stars, based on 5650 article reviews
    Price from $9.99 to $1999.99
    egf - by Bioz Stars, 2020-05
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    97
    Cell Signaling Technology Inc epidermal growth factor egf
    Effect of insect tea on NF-κB, <t>IκB-α,</t> <t>EGF</t> and EGFR protein expression in HCl/ethanol-induced gastric mucosal injury mice. Data are relative to the control group and are presented as the mean + standard deviation (n=7). *P
    Epidermal Growth Factor Egf, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/epidermal growth factor egf/product/Cell Signaling Technology Inc
    Average 97 stars, based on 52 article reviews
    Price from $9.99 to $1999.99
    epidermal growth factor egf - by Bioz Stars, 2020-05
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    Image Search Results


    Npnt regulates Sox2 expression via EGF signaling pathway. ( A ) Ratio of Sox2+ cells among M3H1 cells transfected with Npnt-FL, with or without EGFR siRNA. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. ( B ) qRT-PCR analysis of Sox2 expression in M3H1 cells transfected with Npnt-FL, with or without EGFR siRNA. Gapdh was used as the internal control. ( C ) Ratio of Sox2 cells among M3H1 cells transfected with Npnt-FL, or treated with EGFR inhibitor gefitinib or lapatinib. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. ( D ) qRT-PCR analysis of Sox2 expression in M3H1 cells transfected with Npnt-FL, or treated with EGFR inhibitor gefitinib or lapatinib. Gapdh was used as the internal control. *P

    Journal: Scientific Reports

    Article Title: Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

    doi: 10.1038/srep45181

    Figure Lengend Snippet: Npnt regulates Sox2 expression via EGF signaling pathway. ( A ) Ratio of Sox2+ cells among M3H1 cells transfected with Npnt-FL, with or without EGFR siRNA. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. ( B ) qRT-PCR analysis of Sox2 expression in M3H1 cells transfected with Npnt-FL, with or without EGFR siRNA. Gapdh was used as the internal control. ( C ) Ratio of Sox2 cells among M3H1 cells transfected with Npnt-FL, or treated with EGFR inhibitor gefitinib or lapatinib. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. ( D ) qRT-PCR analysis of Sox2 expression in M3H1 cells transfected with Npnt-FL, or treated with EGFR inhibitor gefitinib or lapatinib. Gapdh was used as the internal control. *P

    Article Snippet: To inhibit EGF signaling, gefitinib (1 μM, Sigma Aldrich) and lapatinib (1 μM, Sigma Aldrich) were applied to M3H1 cells.

    Techniques: Expressing, Transfection, Staining, Quantitative RT-PCR

    scFvE3 is a selective sensor of RhoB activation in HeLa cells. A, CBD-pulldown experiments on nucleotides loaded HeLa cell extracts showing the specificities of the selected scFvs. HeLa cell extracts were loaded with either GDP (1 mM) or GTPγS (100 µM) and incubated with scFvs F7, D10 and E3 fixed on chitin beads. CBD-pulldowns were analyzed by Western blotting using anti-RhoA, anti-RhoB and anti-RhoC antibodies. Total extract used for CBD-pulldown is indicated as input and examined by western blotting with the same antibodies. Western Blot is representative of 4 independent experiments. B, RhoB and RhoA activation were assessed by GST-pulldown (RBD) and CBD-pulldown (E3) experiments with cell lysate from HeLa cells transiently transfected with plasmids expressing Myc-tagged XPLN or GFP under the control of CMV promotor. XPLN was detected by using an anti-c-myc antibody. C, HeLa cells were serum-starved for 24 h and treated with EGF (2.5 ng/mL) for 10 min before lysis then RhoB and RhoA activation were assessed by GST-pulldown (RBD) and CBD-pulldown (E3) experiments. Beads-bound proteins were analyzed by Western blotting using anti-RhoA and anti-RhoB antibodies. Total cell extracts are indicated as input and examined by western blotting with the same antibodies. Western Blots are representatives of 2 independent experiments.

    Journal: PLoS ONE

    Article Title: Generation of a Single Chain Antibody Variable Fragment (scFv) to Sense Selectively RhoB Activation

    doi: 10.1371/journal.pone.0111034

    Figure Lengend Snippet: scFvE3 is a selective sensor of RhoB activation in HeLa cells. A, CBD-pulldown experiments on nucleotides loaded HeLa cell extracts showing the specificities of the selected scFvs. HeLa cell extracts were loaded with either GDP (1 mM) or GTPγS (100 µM) and incubated with scFvs F7, D10 and E3 fixed on chitin beads. CBD-pulldowns were analyzed by Western blotting using anti-RhoA, anti-RhoB and anti-RhoC antibodies. Total extract used for CBD-pulldown is indicated as input and examined by western blotting with the same antibodies. Western Blot is representative of 4 independent experiments. B, RhoB and RhoA activation were assessed by GST-pulldown (RBD) and CBD-pulldown (E3) experiments with cell lysate from HeLa cells transiently transfected with plasmids expressing Myc-tagged XPLN or GFP under the control of CMV promotor. XPLN was detected by using an anti-c-myc antibody. C, HeLa cells were serum-starved for 24 h and treated with EGF (2.5 ng/mL) for 10 min before lysis then RhoB and RhoA activation were assessed by GST-pulldown (RBD) and CBD-pulldown (E3) experiments. Beads-bound proteins were analyzed by Western blotting using anti-RhoA and anti-RhoB antibodies. Total cell extracts are indicated as input and examined by western blotting with the same antibodies. Western Blots are representatives of 2 independent experiments.

    Article Snippet: For EGF stimulation experiments, HeLa cells were cultured in serum free media for 24 h before addition of EGF (Sigma) (2.5 ng/mL) for 10 min.

    Techniques: Activation Assay, Incubation, Western Blot, Transfection, Expressing, Lysis

    PR55γ Is a Regulator of JNK following UV Irradiation (A) U2-OS cells expressing pcDNA-HA-PR55γ were cotransfected with the pooled knockdown (PR55γ KD ) vectors as indicated (A–E) or a control vector. GFP expression serves as a measure of transfection consistency. (B) U2-OS cells were cotransfected with PR55γ KD vectors #1 or #2, pcDNA-PR55γ serves as a positive control. pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. mRNA levels relative to the control are shown as evaluated by quantitative real-time PCR. (C) U2-OS cells were cotransfected with PR55γ KD vectors as indicated (#1 or #2) or control vector. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells expressing pcDNA-HA-PR55γ or pcDNA-HA-PR55γ (Δ) were cotransfected with PR55γ KD vector #2. Protein samples were analyzed by immunoblotting with antibodies targeting HA. (E) U2-OS cells expressing pcDNA-HA-PR55γ, pcDNA-HA- PR55γ(Δ), or a control vector were cotransfected with PR55γ KD vectors #1 or #2. A pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), total JNK (α-JNK), or haemoglutinin (α-HA, reprobe). (F) U2-OS cells expressing PR55γ KD2 vector or a control vector exposed to TNF-α, EGF, NaCl, or insulin for 5 min and incubated for a further 30–60 minutes. pJNK relative to total JNK levels are shown. doi:10.1371/journal.pgen.0030218.g002

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: PR55γ Is a Regulator of JNK following UV Irradiation (A) U2-OS cells expressing pcDNA-HA-PR55γ were cotransfected with the pooled knockdown (PR55γ KD ) vectors as indicated (A–E) or a control vector. GFP expression serves as a measure of transfection consistency. (B) U2-OS cells were cotransfected with PR55γ KD vectors #1 or #2, pcDNA-PR55γ serves as a positive control. pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. mRNA levels relative to the control are shown as evaluated by quantitative real-time PCR. (C) U2-OS cells were cotransfected with PR55γ KD vectors as indicated (#1 or #2) or control vector. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells expressing pcDNA-HA-PR55γ or pcDNA-HA-PR55γ (Δ) were cotransfected with PR55γ KD vector #2. Protein samples were analyzed by immunoblotting with antibodies targeting HA. (E) U2-OS cells expressing pcDNA-HA-PR55γ, pcDNA-HA- PR55γ(Δ), or a control vector were cotransfected with PR55γ KD vectors #1 or #2. A pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), total JNK (α-JNK), or haemoglutinin (α-HA, reprobe). (F) U2-OS cells expressing PR55γ KD2 vector or a control vector exposed to TNF-α, EGF, NaCl, or insulin for 5 min and incubated for a further 30–60 minutes. pJNK relative to total JNK levels are shown. doi:10.1371/journal.pgen.0030218.g002

    Article Snippet: The following agents were used to stimulate cells: 50 ng/ml EGF (Upstate), 10 ng/ml TNF (Sigma), 10 ng/ml Insulin (Sigma), 500 mM NACL, or UVC (254 nm, 100 J/m2 ).

    Techniques: Irradiation, Expressing, Plasmid Preparation, Transfection, Positive Control, shRNA, Real-time Polymerase Chain Reaction, Incubation

    Secreted-form HB-EGF suppresses ΔC-CNIL caused cranial nerve abnormality. (A) The production of biologically active secreted (s) growth factors was confirmed by the detection of activated MAPK after treating HEK-293::HER4(ErbB4) cells with conditioned medium from cultures of sHB-EGF, sNRG1, and sNRG2 expression plasmid-transfected HEK-293T cells. (B–D) ΔC-CNIL and sHB-EGF, ΔC-CNIL and sNRG1, and ΔC-CNIL and sNRG2 expression plasmids, respectively, were electroporated into st-9 chick hindbrains, and nerve fibers were immuno-stained at st-20. Note that only in C and D do abnormal nerve connections (arrows) exist between the Vth (V) and VIIth (VII) ganglia. r3, rhombomere3.

    Journal: Molecular Biology of the Cell

    Article Title: Cornichon-like Protein Facilitates Secretion of HB-EGF and Regulates Proper Development of Cranial Nerves

    doi: 10.1091/mbc.E06-08-0733

    Figure Lengend Snippet: Secreted-form HB-EGF suppresses ΔC-CNIL caused cranial nerve abnormality. (A) The production of biologically active secreted (s) growth factors was confirmed by the detection of activated MAPK after treating HEK-293::HER4(ErbB4) cells with conditioned medium from cultures of sHB-EGF, sNRG1, and sNRG2 expression plasmid-transfected HEK-293T cells. (B–D) ΔC-CNIL and sHB-EGF, ΔC-CNIL and sNRG1, and ΔC-CNIL and sNRG2 expression plasmids, respectively, were electroporated into st-9 chick hindbrains, and nerve fibers were immuno-stained at st-20. Note that only in C and D do abnormal nerve connections (arrows) exist between the Vth (V) and VIIth (VII) ganglia. r3, rhombomere3.

    Article Snippet: After 24 h, the medium was changed to medium without serum, and then the cells were cultured further for 24 h. The conditioned medium was centrifuged at 16,100 rcf for 10 min to remove floating cells, and sHB-EGF was concentrated with a Microcon YM-3 (Millipore, Bedford, MA).

    Techniques: Expressing, Plasmid Preparation, Transfection, Staining

    Tenascin-C modifies the patterns of distribution for filamentous actin, epidermal growth factor receptors, tyrosine-phosphorylated proteins, and vinculin. ( A ) Representative immunofluorescence photomicrographs (from two different experiments) showing distribution patterns for F-actin, EGF-Rs, and tyrosine-phosphorylated (P-Tyr) proteins in SMC cultured in SFM (+/- 50 ng/ml EGF for 30 min) on collagen alone or on TN-C (15 μg/ml) supplemented collagen substrates. Rhodamine-phalloidin staining of SMC on collagen revealed a longitudinal F-actin stress fiber pattern of distribution that was more cortical after treatment with EGF. Immunofluorescent staining for EGF-Rs and tyrosine-phosphorylated proteins was diffuse in SMC cultured on collagen alone and increased modestly after addition of EGF. In contrast, SMC cultured on TN-C–supplemented collagen gels showed high-intensity F-actin staining in regions that often overlapped with clusters of EGF-Rs and tyrosine-phosphorylated proteins. After addition of EGF to TN-C–treated cultures, the levels and distribution of EGF-Rs remained pronounced, and high levels of tyrosine-phosphorylated proteins were evident throughout the cell. ( B ) Representative double immunofluorescence photomicrographs showing codistribution of vinculin, F-actin, EGF-Rs, and tyrosine-phosphorylated (P-Tyr) proteins in SMC cultured in SFM on collagen supplemented with exogenous human TN-C protein. Bars: ( A ) 20 μm; ( B ) 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: Regulation of Tenascin-C, a Vascular Smooth Muscle Cell Survival Factor that Interacts with the ?v?3 Integrin to Promote Epidermal Growth Factor Receptor Phosphorylation and Growth

    doi:

    Figure Lengend Snippet: Tenascin-C modifies the patterns of distribution for filamentous actin, epidermal growth factor receptors, tyrosine-phosphorylated proteins, and vinculin. ( A ) Representative immunofluorescence photomicrographs (from two different experiments) showing distribution patterns for F-actin, EGF-Rs, and tyrosine-phosphorylated (P-Tyr) proteins in SMC cultured in SFM (+/- 50 ng/ml EGF for 30 min) on collagen alone or on TN-C (15 μg/ml) supplemented collagen substrates. Rhodamine-phalloidin staining of SMC on collagen revealed a longitudinal F-actin stress fiber pattern of distribution that was more cortical after treatment with EGF. Immunofluorescent staining for EGF-Rs and tyrosine-phosphorylated proteins was diffuse in SMC cultured on collagen alone and increased modestly after addition of EGF. In contrast, SMC cultured on TN-C–supplemented collagen gels showed high-intensity F-actin staining in regions that often overlapped with clusters of EGF-Rs and tyrosine-phosphorylated proteins. After addition of EGF to TN-C–treated cultures, the levels and distribution of EGF-Rs remained pronounced, and high levels of tyrosine-phosphorylated proteins were evident throughout the cell. ( B ) Representative double immunofluorescence photomicrographs showing codistribution of vinculin, F-actin, EGF-Rs, and tyrosine-phosphorylated (P-Tyr) proteins in SMC cultured in SFM on collagen supplemented with exogenous human TN-C protein. Bars: ( A ) 20 μm; ( B ) 5 μm.

    Article Snippet: To detect EGF-Rs, tyrosine-phosphorylated proteins, and vinculin, SMCs were fixed in 2% paraformaldehyde ( Sigma Chemical Co. ) for 10 min at room temperature and rinsed in PBS containing 0.1 M glycine for 30 min. For tyrosine-phosphorylated proteins and vinculin, SMCs were permeabilized with 0.2% Triton X-100 in PBS for 5 min and washed three more times.

    Techniques: Immunofluorescence, Cell Culture, Staining

    The role of β 3 integrins in tenascin-C–dependent epidermal growth factor receptor clustering and ligand-dependent receptor activation. ( A ) Representative immunofluorescence photomicrographs for EGF-R in SMCs cultured on TN-C (15 μg/ ml) supplemented collagen gels in the presence of control IgG or with an anti–β 3 integrin antibody that prevented the formation of EGF-R clusters. ( B ) Effect of blocking β 3 integrins on ligand-dependent EGF-R tyrosine phosphorylation in SMCs cultured on collagen gels supplemented with TN-C. Western immunoblot (representative of two different studies) of membrane preparations from SMCs treated with control IgG, with anti–β 3 integrin antisera on TN-C–supplemented collagen in SFM alone, or in SFM with EGF (50 ng/ml) for 10 and 30 min. Epidermal growth factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions and were analyzed for evidence of tyrosine phosphorylation using Western immunoblotting. Note that the anti–β 3 integrin antibody prevents the transient increase in EGF-R tyrosine phosphorylation observed in TN-treated cultures after addition of EGF at 10 min. Bar, 17 μm.

    Journal: The Journal of Cell Biology

    Article Title: Regulation of Tenascin-C, a Vascular Smooth Muscle Cell Survival Factor that Interacts with the ?v?3 Integrin to Promote Epidermal Growth Factor Receptor Phosphorylation and Growth

    doi:

    Figure Lengend Snippet: The role of β 3 integrins in tenascin-C–dependent epidermal growth factor receptor clustering and ligand-dependent receptor activation. ( A ) Representative immunofluorescence photomicrographs for EGF-R in SMCs cultured on TN-C (15 μg/ ml) supplemented collagen gels in the presence of control IgG or with an anti–β 3 integrin antibody that prevented the formation of EGF-R clusters. ( B ) Effect of blocking β 3 integrins on ligand-dependent EGF-R tyrosine phosphorylation in SMCs cultured on collagen gels supplemented with TN-C. Western immunoblot (representative of two different studies) of membrane preparations from SMCs treated with control IgG, with anti–β 3 integrin antisera on TN-C–supplemented collagen in SFM alone, or in SFM with EGF (50 ng/ml) for 10 and 30 min. Epidermal growth factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions and were analyzed for evidence of tyrosine phosphorylation using Western immunoblotting. Note that the anti–β 3 integrin antibody prevents the transient increase in EGF-R tyrosine phosphorylation observed in TN-treated cultures after addition of EGF at 10 min. Bar, 17 μm.

    Article Snippet: To detect EGF-Rs, tyrosine-phosphorylated proteins, and vinculin, SMCs were fixed in 2% paraformaldehyde ( Sigma Chemical Co. ) for 10 min at room temperature and rinsed in PBS containing 0.1 M glycine for 30 min. For tyrosine-phosphorylated proteins and vinculin, SMCs were permeabilized with 0.2% Triton X-100 in PBS for 5 min and washed three more times.

    Techniques: Activation Assay, Immunofluorescence, Cell Culture, Blocking Assay, Western Blot, Immunoprecipitation

    Regulation of tenascin-C protein expression by matrix metalloproteinases and β 3 integrins. ( A ) Immunoprecipitation of TN-C protein from [ 35 S]methionine/cysteine-labeled SMCs cultured on native type I collagen gels in serum-free control medium with 50 ng/ml EGF and 0.4% DMSO, or in control medium supplemented with 1 or 2 μM of GM6001, an MMP inhibitor. Inhibition of MMP activity with GM6001 leads to a marked decrease in TN-C protein synthesis. Positions of molecular mass standards in kD are indicated on the left. ( B ) Western immunoblotting for TN-C protein from SMCs cultured in SFM with 50 ng/ml EGF on native and heat-denatured type I collagen gels. A Western immunoblot shows that expression of two TN-C isoforms of apparent molecular masses 220 and 180 kD is increased in cells cultured on denatured collagen. ( C ) Western immunoblot of TN-C protein from SMCs cultured on heat-denatured type I collagen gels in control medium supplemented with either 0.4% DMSO or 2 μM of GM6001. Inhibition of MMP activity with GM6001 had no effect on TN-C protein expression. ( D ) Effect of β 3 integrin blockade on TN-C protein expression on heat-denatured collagen. Immunoprecipitation of TN-C protein from [ 35 S]methionine/cysteine- labeled SMCs cultured on native type I collagen gels in the presence of control IgG or with anti-β 3 antibody shows that blocking β 3 integrins inhibits TN-C protein expression on native type I collagen. ( E ) Effect of β 3 integrin blockade on TN-C protein expression on heat-denatured collagen. Western immunoblot analysis of lysates derived from cells cultured on heat-denatured collagen in the presence of control IgG, or with anti-β 3 integrin antibody shows that blocking β 3 integrins inhibits TN-C protein expression on denatured collagen. Arrows indicate the presence of the 220-kD ( upper band ) and 180-kD ( lower band ) TN-C isoforms.

    Journal: The Journal of Cell Biology

    Article Title: Regulation of Tenascin-C, a Vascular Smooth Muscle Cell Survival Factor that Interacts with the ?v?3 Integrin to Promote Epidermal Growth Factor Receptor Phosphorylation and Growth

    doi:

    Figure Lengend Snippet: Regulation of tenascin-C protein expression by matrix metalloproteinases and β 3 integrins. ( A ) Immunoprecipitation of TN-C protein from [ 35 S]methionine/cysteine-labeled SMCs cultured on native type I collagen gels in serum-free control medium with 50 ng/ml EGF and 0.4% DMSO, or in control medium supplemented with 1 or 2 μM of GM6001, an MMP inhibitor. Inhibition of MMP activity with GM6001 leads to a marked decrease in TN-C protein synthesis. Positions of molecular mass standards in kD are indicated on the left. ( B ) Western immunoblotting for TN-C protein from SMCs cultured in SFM with 50 ng/ml EGF on native and heat-denatured type I collagen gels. A Western immunoblot shows that expression of two TN-C isoforms of apparent molecular masses 220 and 180 kD is increased in cells cultured on denatured collagen. ( C ) Western immunoblot of TN-C protein from SMCs cultured on heat-denatured type I collagen gels in control medium supplemented with either 0.4% DMSO or 2 μM of GM6001. Inhibition of MMP activity with GM6001 had no effect on TN-C protein expression. ( D ) Effect of β 3 integrin blockade on TN-C protein expression on heat-denatured collagen. Immunoprecipitation of TN-C protein from [ 35 S]methionine/cysteine- labeled SMCs cultured on native type I collagen gels in the presence of control IgG or with anti-β 3 antibody shows that blocking β 3 integrins inhibits TN-C protein expression on native type I collagen. ( E ) Effect of β 3 integrin blockade on TN-C protein expression on heat-denatured collagen. Western immunoblot analysis of lysates derived from cells cultured on heat-denatured collagen in the presence of control IgG, or with anti-β 3 integrin antibody shows that blocking β 3 integrins inhibits TN-C protein expression on denatured collagen. Arrows indicate the presence of the 220-kD ( upper band ) and 180-kD ( lower band ) TN-C isoforms.

    Article Snippet: To detect EGF-Rs, tyrosine-phosphorylated proteins, and vinculin, SMCs were fixed in 2% paraformaldehyde ( Sigma Chemical Co. ) for 10 min at room temperature and rinsed in PBS containing 0.1 M glycine for 30 min. For tyrosine-phosphorylated proteins and vinculin, SMCs were permeabilized with 0.2% Triton X-100 in PBS for 5 min and washed three more times.

    Techniques: Expressing, Immunoprecipitation, Labeling, Cell Culture, Inhibition, Activity Assay, Western Blot, Blocking Assay, Derivative Assay

    Effect of blocking α v β 3 integrins on tenascin- C–dependent smooth muscle cell morphology, attachment efficiency, and survival. ( A ) Representative phase contrast photomicrographs of SMCs plated in SFM on collagen and TN-C–supplemented collagen gels with control IgG (15 μg/ml) or with an anti–α v β 3 integrin antisera (LM609; 15 μg/ml). IgG and LM609 treatment had no effect on the stellate morphology produced by the collagen substrate. In contrast, the more elongated SMC morphology observed on control-treated TN-C–enriched substrates was abrogated by inclusion of LM609, which prevented cells from spreading, resulting in a rounded morphology. ( B ) Effect of LM609 antisera on SMC attachment to TN-C–supplemented type I collagen gels. By 6 h after plating, no significant differences in attachment efficiency were noted between cells plated in SFM on TN-C–supplemented collagen gels in either the presence of control IgG or LM609 antisera. ( C ) Effect of blocking SMC α v β 3 integrins with LM609 antisera on EGF-dependent SMC cell growth on TN-C–supplemented collagen gels. Control and LM609 treatment produced no differences in SMC number after culture for 48 h on collagen gels in SFM with 50 ng/ml EGF. The significant ( P

    Journal: The Journal of Cell Biology

    Article Title: Regulation of Tenascin-C, a Vascular Smooth Muscle Cell Survival Factor that Interacts with the ?v?3 Integrin to Promote Epidermal Growth Factor Receptor Phosphorylation and Growth

    doi:

    Figure Lengend Snippet: Effect of blocking α v β 3 integrins on tenascin- C–dependent smooth muscle cell morphology, attachment efficiency, and survival. ( A ) Representative phase contrast photomicrographs of SMCs plated in SFM on collagen and TN-C–supplemented collagen gels with control IgG (15 μg/ml) or with an anti–α v β 3 integrin antisera (LM609; 15 μg/ml). IgG and LM609 treatment had no effect on the stellate morphology produced by the collagen substrate. In contrast, the more elongated SMC morphology observed on control-treated TN-C–enriched substrates was abrogated by inclusion of LM609, which prevented cells from spreading, resulting in a rounded morphology. ( B ) Effect of LM609 antisera on SMC attachment to TN-C–supplemented type I collagen gels. By 6 h after plating, no significant differences in attachment efficiency were noted between cells plated in SFM on TN-C–supplemented collagen gels in either the presence of control IgG or LM609 antisera. ( C ) Effect of blocking SMC α v β 3 integrins with LM609 antisera on EGF-dependent SMC cell growth on TN-C–supplemented collagen gels. Control and LM609 treatment produced no differences in SMC number after culture for 48 h on collagen gels in SFM with 50 ng/ml EGF. The significant ( P

    Article Snippet: To detect EGF-Rs, tyrosine-phosphorylated proteins, and vinculin, SMCs were fixed in 2% paraformaldehyde ( Sigma Chemical Co. ) for 10 min at room temperature and rinsed in PBS containing 0.1 M glycine for 30 min. For tyrosine-phosphorylated proteins and vinculin, SMCs were permeabilized with 0.2% Triton X-100 in PBS for 5 min and washed three more times.

    Techniques: Blocking Assay, Produced

    Tenascin-C potentiates ligand-dependent epidermal growth factor-receptor tyrosine phosphorylation. ( A ) Effect of TN-C on EGF-R protein synthesis. Representative autoradiograph (from two different experiments) showing immunoprecipitation of EGF-R protein from [ 35 S]methionine-labeled SMCs cultured on native type I collagen gels (+/− 15 μg/ml TN-C) in SFM indicates that TN-C has no effect on the levels of EGF-R protein synthesis compared to SMCs cultured on collagen alone. ( B ) Effect of TN-C on tyrosine phosphorylation in SMCs cultured on collagen and TN-C–supplemented gels in SFM, or after addition of EGF (50 ng/ml) for 10 and 30 min. A Western immunoblot (representative of two different studies) was performed on membrane-enriched fractions (15 μg per sample) using an antibody against tyrosine-phosphorylated proteins. Note that relative to SMCs cultured on collagen alone, contact with exogenous TN-C led to an increase in basal levels of tyrosine phosphorylation. In response to EGF, qualitative and quantitative increases in tyrosine-phosphorylated proteins were observed on both collagen and TN-C, with changes being more pronounced on TN-C–enriched gels, including species at 170 and 125 kD. Positions of molecular mass standards in kD are indicated on the right. The 170-kD species may represent the EGF receptor, and the 125-kD species the focal adhesion kinase. ( C ) The 170-kD tyrosine-phosphorylated protein shown in B represents the EGF-R. Epidermal growth factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions in cells cultured as in B. Epidermal growth factor immunoprecipitates were analyzed for evidence of tyrosine phosphorylation using Western immunoblotting with an antiphosphotyrosine antibody. After addition of EGF at 10 min, a transient increase in the levels of tyrosine-phosphorylated EGF-Rs occurs on collagen and TN-C–supplemented gels, with greater levels observed on TN-C–supplemented collagen substrates.

    Journal: The Journal of Cell Biology

    Article Title: Regulation of Tenascin-C, a Vascular Smooth Muscle Cell Survival Factor that Interacts with the ?v?3 Integrin to Promote Epidermal Growth Factor Receptor Phosphorylation and Growth

    doi:

    Figure Lengend Snippet: Tenascin-C potentiates ligand-dependent epidermal growth factor-receptor tyrosine phosphorylation. ( A ) Effect of TN-C on EGF-R protein synthesis. Representative autoradiograph (from two different experiments) showing immunoprecipitation of EGF-R protein from [ 35 S]methionine-labeled SMCs cultured on native type I collagen gels (+/− 15 μg/ml TN-C) in SFM indicates that TN-C has no effect on the levels of EGF-R protein synthesis compared to SMCs cultured on collagen alone. ( B ) Effect of TN-C on tyrosine phosphorylation in SMCs cultured on collagen and TN-C–supplemented gels in SFM, or after addition of EGF (50 ng/ml) for 10 and 30 min. A Western immunoblot (representative of two different studies) was performed on membrane-enriched fractions (15 μg per sample) using an antibody against tyrosine-phosphorylated proteins. Note that relative to SMCs cultured on collagen alone, contact with exogenous TN-C led to an increase in basal levels of tyrosine phosphorylation. In response to EGF, qualitative and quantitative increases in tyrosine-phosphorylated proteins were observed on both collagen and TN-C, with changes being more pronounced on TN-C–enriched gels, including species at 170 and 125 kD. Positions of molecular mass standards in kD are indicated on the right. The 170-kD species may represent the EGF receptor, and the 125-kD species the focal adhesion kinase. ( C ) The 170-kD tyrosine-phosphorylated protein shown in B represents the EGF-R. Epidermal growth factor receptors were immunoprecipitated from 15 μg of membrane-enriched SMC fractions in cells cultured as in B. Epidermal growth factor immunoprecipitates were analyzed for evidence of tyrosine phosphorylation using Western immunoblotting with an antiphosphotyrosine antibody. After addition of EGF at 10 min, a transient increase in the levels of tyrosine-phosphorylated EGF-Rs occurs on collagen and TN-C–supplemented gels, with greater levels observed on TN-C–supplemented collagen substrates.

    Article Snippet: To detect EGF-Rs, tyrosine-phosphorylated proteins, and vinculin, SMCs were fixed in 2% paraformaldehyde ( Sigma Chemical Co. ) for 10 min at room temperature and rinsed in PBS containing 0.1 M glycine for 30 min. For tyrosine-phosphorylated proteins and vinculin, SMCs were permeabilized with 0.2% Triton X-100 in PBS for 5 min and washed three more times.

    Techniques: Autoradiography, Immunoprecipitation, Labeling, Cell Culture, Western Blot

    Tenascin-C and matrix metalloproteinase-2 (MMP-2) expression in vascular smooth muscle cells. ( A ) Representative photomicrograph showing SMC morphology on attached type I collagen gels and on collagen gels that have been released for 2 h into the culture medium. ( B ) Autoradiograph (representative of two different experiments) showing immunoprecipitated TN protein from [ 35 S]methionine/cysteine-labeled SMC lysates harvested from attached and floating type I collagen gels in SFM with 50 ng/ml EGF at 24 h. Synthesis of two TN-C protein isoforms of apparent molecular masses 220 and 180 kD, is suppressed in cells cultured on floating collagen. ( C ) Gelatin zymography was used to examine the levels and activity of gelatinases present in conditioned medium (CM) harvested from SMCs cultured on attached and floating collagen. Fresh SFM with 50 ng/ml EGF was added for 4 h before its collection at 4, 8, 12, and 24 h. Equal volumes of concentrated CM were then analyzed by gelatin substrate zymography in nonreducing 10% polyacrylamide gels containing 0.1% gelatin. A zymogram (representative of three different experiments) shows that on attached and floating collagen, SMCs secrete gelatinases with apparent molecular masses of 68 and 57 kD, and that on floating collagen, the activity is lower. ( D ) Densitometry of the 68- and 57-kD gelatinases shown in C. ( E ) Western immunoblot for the latent form of MMP-2 in conditioned medium harvested at 24 h from SMCs cultured on attached and floating collagen shows that expression of the 72-kD proform of this enzyme (equivalent to the 68-kD species under nonreducing conditions on the zymogram) is suppressed under floating conditions. Bar, 120 μm.

    Journal: The Journal of Cell Biology

    Article Title: Regulation of Tenascin-C, a Vascular Smooth Muscle Cell Survival Factor that Interacts with the ?v?3 Integrin to Promote Epidermal Growth Factor Receptor Phosphorylation and Growth

    doi:

    Figure Lengend Snippet: Tenascin-C and matrix metalloproteinase-2 (MMP-2) expression in vascular smooth muscle cells. ( A ) Representative photomicrograph showing SMC morphology on attached type I collagen gels and on collagen gels that have been released for 2 h into the culture medium. ( B ) Autoradiograph (representative of two different experiments) showing immunoprecipitated TN protein from [ 35 S]methionine/cysteine-labeled SMC lysates harvested from attached and floating type I collagen gels in SFM with 50 ng/ml EGF at 24 h. Synthesis of two TN-C protein isoforms of apparent molecular masses 220 and 180 kD, is suppressed in cells cultured on floating collagen. ( C ) Gelatin zymography was used to examine the levels and activity of gelatinases present in conditioned medium (CM) harvested from SMCs cultured on attached and floating collagen. Fresh SFM with 50 ng/ml EGF was added for 4 h before its collection at 4, 8, 12, and 24 h. Equal volumes of concentrated CM were then analyzed by gelatin substrate zymography in nonreducing 10% polyacrylamide gels containing 0.1% gelatin. A zymogram (representative of three different experiments) shows that on attached and floating collagen, SMCs secrete gelatinases with apparent molecular masses of 68 and 57 kD, and that on floating collagen, the activity is lower. ( D ) Densitometry of the 68- and 57-kD gelatinases shown in C. ( E ) Western immunoblot for the latent form of MMP-2 in conditioned medium harvested at 24 h from SMCs cultured on attached and floating collagen shows that expression of the 72-kD proform of this enzyme (equivalent to the 68-kD species under nonreducing conditions on the zymogram) is suppressed under floating conditions. Bar, 120 μm.

    Article Snippet: To detect EGF-Rs, tyrosine-phosphorylated proteins, and vinculin, SMCs were fixed in 2% paraformaldehyde ( Sigma Chemical Co. ) for 10 min at room temperature and rinsed in PBS containing 0.1 M glycine for 30 min. For tyrosine-phosphorylated proteins and vinculin, SMCs were permeabilized with 0.2% Triton X-100 in PBS for 5 min and washed three more times.

    Techniques: Expressing, Autoradiography, Immunoprecipitation, Labeling, Cell Culture, Zymography, Activity Assay, Western Blot

    In vitro permeation through the skin PAMPA system. Time-course of cumulative permeated concentrations of ( A ) rhEGF and rLMWP-EGF; ( B ) rhIGF-I and rLMWP-IGF-I; and ( C ) rhPDGF-A and rLMWP-PDGF-A, after 4, 6, 8, and 24 hours. Notes: Each value represents the mean ± standard deviation (n=6). * P

    Journal: International Journal of Nanomedicine

    Article Title: Enhanced regenerative healing efficacy of a highly skin-permeable growth factor nanocomplex in a full-thickness excisional mouse wound model

    doi: 10.2147/IJN.S68399

    Figure Lengend Snippet: In vitro permeation through the skin PAMPA system. Time-course of cumulative permeated concentrations of ( A ) rhEGF and rLMWP-EGF; ( B ) rhIGF-I and rLMWP-IGF-I; and ( C ) rhPDGF-A and rLMWP-PDGF-A, after 4, 6, 8, and 24 hours. Notes: Each value represents the mean ± standard deviation (n=6). * P

    Article Snippet: The supernatant, containing rLMWP-EGF, rLMWP-IGF-I, or rLMWP-PDGF-A, was collected by centrifugation (12,000× g , 10 minutes, 4°C) and filtered through a 0.45 μm membrane (EMD Millipore, Billerica, MA, USA) before further analysis.

    Techniques: In Vitro, PAMPA Assay, Standard Deviation

    Effect of an LMWP-conjugated growth factor nanocomplex gel formulation on wound area reduction in the full-thickness excisional wound model. ( A ) Measurements of the wound area measured over 9 days. ( B ) Representative photographs of wounds treated with vehicle (Poloxamer 188, 6% [w/v]) alone or with vehicle containing one of the following: 10 μg/mL rhEGF; 10 μg/mL rLMWP-EGF; 10 μg/mL rhIGF-I; 10 μg/mL rLMWP-IGF-I; 10 μg/mL rhPDGF-A; 10 μg/mL rLMWP-PDGF-A; native growth factor combination (10 μg/mL each of rhEGF, rhIGF-I, and rhPDGF-A); LMWP-conjugated growth factor combination (10 μg/mL each of rLMWP-EGF, rLMWP-IGF-I, and rLMWP-PDGF-A); native growth factor nanocomplex (10 μg/mL each of rhEGF, rhIGF-I, and rhPDGF-A, with 100 μg/mL LMWH); or LMWP-conjugated growth factor nanocomplex (10 μg/mL each of rLMWP-EGF, rLMWP-IGF-I, and rLMWP-PDGF-A, with 100 μg/mL LMWH). Notes: Each value represents the mean ± standard deviation (n=10). * P

    Journal: International Journal of Nanomedicine

    Article Title: Enhanced regenerative healing efficacy of a highly skin-permeable growth factor nanocomplex in a full-thickness excisional mouse wound model

    doi: 10.2147/IJN.S68399

    Figure Lengend Snippet: Effect of an LMWP-conjugated growth factor nanocomplex gel formulation on wound area reduction in the full-thickness excisional wound model. ( A ) Measurements of the wound area measured over 9 days. ( B ) Representative photographs of wounds treated with vehicle (Poloxamer 188, 6% [w/v]) alone or with vehicle containing one of the following: 10 μg/mL rhEGF; 10 μg/mL rLMWP-EGF; 10 μg/mL rhIGF-I; 10 μg/mL rLMWP-IGF-I; 10 μg/mL rhPDGF-A; 10 μg/mL rLMWP-PDGF-A; native growth factor combination (10 μg/mL each of rhEGF, rhIGF-I, and rhPDGF-A); LMWP-conjugated growth factor combination (10 μg/mL each of rLMWP-EGF, rLMWP-IGF-I, and rLMWP-PDGF-A); native growth factor nanocomplex (10 μg/mL each of rhEGF, rhIGF-I, and rhPDGF-A, with 100 μg/mL LMWH); or LMWP-conjugated growth factor nanocomplex (10 μg/mL each of rLMWP-EGF, rLMWP-IGF-I, and rLMWP-PDGF-A, with 100 μg/mL LMWH). Notes: Each value represents the mean ± standard deviation (n=10). * P

    Article Snippet: The supernatant, containing rLMWP-EGF, rLMWP-IGF-I, or rLMWP-PDGF-A, was collected by centrifugation (12,000× g , 10 minutes, 4°C) and filtered through a 0.45 μm membrane (EMD Millipore, Billerica, MA, USA) before further analysis.

    Techniques: Standard Deviation

    ( A ) SDS-PAGE analysis of native growth factors and purified LMWP-conjugated growth factors: Markers (M); rhEGF (lane 1); rLMWP-EGF (lane 2); rhIGF-I (lane 3); rLMWP-IGF-I (lane 4); rhPDGF-A (lane 5); and rLMWP-PDGF-A (lane 6). ( B ) The actual molecular weights of rLMWP-EGF, rLMWP-IGF-I, and rLMWP-PDGF-A, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. ( C ) Representative chromatograms for N-terminal amino acid sequences of rLMWP-IGF-I and rLMWP-PDGF-A; residues 2, 6, 14, and 16 of rLMWP-IGF-I and rLMWP-PDGF-A. Abbreviations: EGF, epidermal growth factor; IGF-I, insulin-like growth factor 1; LMWP, low-molecular-weight protamine; PDGF-A, platelet-derived growth factor A ligand; r, recombinant; rh, recombinant human; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

    Journal: International Journal of Nanomedicine

    Article Title: Enhanced regenerative healing efficacy of a highly skin-permeable growth factor nanocomplex in a full-thickness excisional mouse wound model

    doi: 10.2147/IJN.S68399

    Figure Lengend Snippet: ( A ) SDS-PAGE analysis of native growth factors and purified LMWP-conjugated growth factors: Markers (M); rhEGF (lane 1); rLMWP-EGF (lane 2); rhIGF-I (lane 3); rLMWP-IGF-I (lane 4); rhPDGF-A (lane 5); and rLMWP-PDGF-A (lane 6). ( B ) The actual molecular weights of rLMWP-EGF, rLMWP-IGF-I, and rLMWP-PDGF-A, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. ( C ) Representative chromatograms for N-terminal amino acid sequences of rLMWP-IGF-I and rLMWP-PDGF-A; residues 2, 6, 14, and 16 of rLMWP-IGF-I and rLMWP-PDGF-A. Abbreviations: EGF, epidermal growth factor; IGF-I, insulin-like growth factor 1; LMWP, low-molecular-weight protamine; PDGF-A, platelet-derived growth factor A ligand; r, recombinant; rh, recombinant human; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

    Article Snippet: The supernatant, containing rLMWP-EGF, rLMWP-IGF-I, or rLMWP-PDGF-A, was collected by centrifugation (12,000× g , 10 minutes, 4°C) and filtered through a 0.45 μm membrane (EMD Millipore, Billerica, MA, USA) before further analysis.

    Techniques: SDS Page, Purification, Mass Spectrometry, Molecular Weight, Derivative Assay, Recombinant, Polyacrylamide Gel Electrophoresis

    Loss of PTEN delays EGFR transport from early to late endosomes. ( a ) Serum-starved control or PTEN shRNA-transduced HeLa cells were pulsed with alexa Flour 647-conjugated EGF (100 ng ml −1 ) for 5 min. Cells fixed at different indicated times were imaged using confocal microscope after co-staining with antibodies against EEA1. Scale bar, 10 μm. ( b ) EGF-EEA1 co-localization in control and PTEN shRNA cells at different times after EGF addition was analysed by Manders method of pixel intensity correlation measurements using Image J/Fiji-Coloc2 plugin. Error bars indicate s.d. ( n =100 cells for each time point from three independent experiments), * P

    Journal: Nature Communications

    Article Title: PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7

    doi: 10.1038/ncomms10689

    Figure Lengend Snippet: Loss of PTEN delays EGFR transport from early to late endosomes. ( a ) Serum-starved control or PTEN shRNA-transduced HeLa cells were pulsed with alexa Flour 647-conjugated EGF (100 ng ml −1 ) for 5 min. Cells fixed at different indicated times were imaged using confocal microscope after co-staining with antibodies against EEA1. Scale bar, 10 μm. ( b ) EGF-EEA1 co-localization in control and PTEN shRNA cells at different times after EGF addition was analysed by Manders method of pixel intensity correlation measurements using Image J/Fiji-Coloc2 plugin. Error bars indicate s.d. ( n =100 cells for each time point from three independent experiments), * P

    Article Snippet: Alexa flour-647-conjugated EGF (Invitrogen), unlabelled EGF, GDPβS, GTPγS (all from Sigma) were used.

    Techniques: shRNA, Microscopy, Staining

    Causal network analysis, the identification and validation of pro-invasive growth-factors in the invasion signature of fibroblasts. A heatmap that indicates a ranking of physiological upstream regulators (filtered for ‘growth factors’) according to their p-values for gene expression data at 72 and 96 hours upon invasion ( a ). The predicted activated upstream regulators were functionally confirmed in vitro by a 3D invasion assay. Therefore, the invasive capacity of MLg fibroblasts was assessed by software-based quantification of the number of fibroblast nuclei (stained with DAPI) within and on top of the collagen matrix of 3D reconstructed confocal z-stacks. Fibroblasts were treated and left to invade the 3D collagen matrices for 48 hours. TGFβ1 (1 and 5 ng/ml), FGF2 (10 and 50 ng/ml), PDGF-BB (5 and 25 ng/ml) and EGF (10 and 50 ng/ml) were tested in the invasion assay ( b - e ). The data shown represent mean values (s.d.) from three to five independent experiment (n = 3–5) including five technical replicates each. (n.t. = non-treated). Statistical analysis: One way ANOVA with Dunnett’s multiple comparison test. *p

    Journal: Scientific Reports

    Article Title: Validated prediction of pro-invasive growth factors using a transcriptome-wide invasion signature derived from a complex 3D invasion assay

    doi: 10.1038/srep12673

    Figure Lengend Snippet: Causal network analysis, the identification and validation of pro-invasive growth-factors in the invasion signature of fibroblasts. A heatmap that indicates a ranking of physiological upstream regulators (filtered for ‘growth factors’) according to their p-values for gene expression data at 72 and 96 hours upon invasion ( a ). The predicted activated upstream regulators were functionally confirmed in vitro by a 3D invasion assay. Therefore, the invasive capacity of MLg fibroblasts was assessed by software-based quantification of the number of fibroblast nuclei (stained with DAPI) within and on top of the collagen matrix of 3D reconstructed confocal z-stacks. Fibroblasts were treated and left to invade the 3D collagen matrices for 48 hours. TGFβ1 (1 and 5 ng/ml), FGF2 (10 and 50 ng/ml), PDGF-BB (5 and 25 ng/ml) and EGF (10 and 50 ng/ml) were tested in the invasion assay ( b - e ). The data shown represent mean values (s.d.) from three to five independent experiment (n = 3–5) including five technical replicates each. (n.t. = non-treated). Statistical analysis: One way ANOVA with Dunnett’s multiple comparison test. *p

    Article Snippet: Treatment of cells with recombinant epidermal growth factor (EGF) (Sigma), transforming growth factor beta-1 (TGFβ1) (R & D, Minneapolis, MN, USA)), fibroblast growth factor (FGF) 2 (R & D) or platelet derived growth factor (PDGF) BB (life technologies; Carlsbad, USA) was accomplished by treating cells with 10–50 ng/ml EGF and FGF2, 1–5 ng/ml TGFβ1, or 5–25 ng/ml PDGF-BB 24 hours after plating, and culturing them up to 72 hours in total.

    Techniques: Expressing, In Vitro, Invasion Assay, Software, Staining

    (A) Cyclopamine inhibits expression of PTCH and GLI1 RNA in LNCaP C4-2B cells: control cells (lanes 1 and 3); 24 hrs 14 nM cyclopamine treatment (lanes 2 and 4). Relative expression determined by QPCR (error bars show standard deviation). (B) Immunoblot showing inhibitory effect of 168 nM gefitinib on EGFR phosphorylation following EGF treatment in AIPC cells (lane 1: control; lane 2: 24 hr gefitinib; lane 3: 48 hr gefitinib). (C) Immunoblot showing inhibitory effect of 102 nM lapatinib on EGFR phosphorylation following EGF treatment in AIPC cells (lane 1: control; lane 2: 24 hr lapatinib; lane 3: 48 hr lapatinib).

    Journal: Cancer Cell International

    Article Title: Inhibition of androgen-independent prostate cancer cell growth is enhanced by combination therapy targeting Hedgehog and ErbB signalling

    doi: 10.1186/1475-2867-8-3

    Figure Lengend Snippet: (A) Cyclopamine inhibits expression of PTCH and GLI1 RNA in LNCaP C4-2B cells: control cells (lanes 1 and 3); 24 hrs 14 nM cyclopamine treatment (lanes 2 and 4). Relative expression determined by QPCR (error bars show standard deviation). (B) Immunoblot showing inhibitory effect of 168 nM gefitinib on EGFR phosphorylation following EGF treatment in AIPC cells (lane 1: control; lane 2: 24 hr gefitinib; lane 3: 48 hr gefitinib). (C) Immunoblot showing inhibitory effect of 102 nM lapatinib on EGFR phosphorylation following EGF treatment in AIPC cells (lane 1: control; lane 2: 24 hr lapatinib; lane 3: 48 hr lapatinib).

    Article Snippet: Cells were treated with EGF (Sigma), cyclopamine (Sigma), gefitinib (AstraZenica) and lapatinib (Glaxo-Smithkline) as detailed.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    Effect of insect tea on NF-κB, IκB-α, EGF and EGFR protein expression in HCl/ethanol-induced gastric mucosal injury mice. Data are relative to the control group and are presented as the mean + standard deviation (n=7). *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Insect tea attenuates hydrochloric acid and ethanol-induced mice acute gastric injury

    doi: 10.3892/etm.2017.5181

    Figure Lengend Snippet: Effect of insect tea on NF-κB, IκB-α, EGF and EGFR protein expression in HCl/ethanol-induced gastric mucosal injury mice. Data are relative to the control group and are presented as the mean + standard deviation (n=7). *P

    Article Snippet: The membrane was blocked with 10% skimmed milk at 4°C for 8 h. Blots were subsequently incubated with antibodies against nuclear factor (NF)-κB (#8214), inhibitor of NF-κB (IκB-α) (#8219), epidermal growth factor (EGF) (#2237), EGF receptor (EGFR) (#11862), endothelial nitric oxide synthase (eNOS) (#32027), neuronal NOS (nNOS) (#4231), inducible NOS (iNOS) (#13120), Mn-SOD (#13141), Cu/Zn-SOD (#4266), catalase (CAT) (#14097) and β-actin (#12262) (all 1:5,000; all Cell Signaling Technology, Inc., Danvers, MA, USA) for 4 h at 4°C.

    Techniques: Expressing, Mouse Assay, Standard Deviation

    EGF induced morphological changes in cells with Tensin4 up-regulation SMMC-7721, LO2, PLC and BEL7402 cells were treated with EGF at a concentration of 40ng/ml for 24 hours. The cells were fixed and counterstained with phalloidin and DAPI for F-actin and cell nuclei. Scale bar: 10 μm.

    Journal: Oncotarget

    Article Title: Tensin4 is up-regulated by EGF-induced ERK1/2 activity and promotes cell proliferation and migration in hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: EGF induced morphological changes in cells with Tensin4 up-regulation SMMC-7721, LO2, PLC and BEL7402 cells were treated with EGF at a concentration of 40ng/ml for 24 hours. The cells were fixed and counterstained with phalloidin and DAPI for F-actin and cell nuclei. Scale bar: 10 μm.

    Article Snippet: Epidermal growth factor (EGF) and inhibitor treatment 1.5×105 cells were seeded overnight in 12-well format and stimulated with 40ng/ml EGF (Cell Signaling Technology, Beverly, CA) in full growth medium for the indicated duration.

    Techniques: Planar Chromatography, Concentration Assay

    EGF-induced Tensin4 up-regulation required persistent ERK activation underlying ERK nuclear translocation A. SMMC-7721 cells were treated with EGF for the indicated durations and then released by washing twice with PBS followed by incubation for up to 8 hours. The cell lysates were then collected and subjected to Western blotting. B. SMMC-7721 and LO2 cells were induced with EGF treatment plus pre-treatment or post-treatment with the MEK inhibitor U0126 at the indicated time points. All the samples were collected at 360 minutes (6 hours) and subjected to Western blotting for the indicated proteins. C. PLC, SMMC-7721 and BEL7402 cells with different basal Tensin4 expression level were treated with EGF for 24 hours. The cell lysates were then collected and subjected to Western blotting. The arrowhead highlights the induction of Tensin4 in BEL7402 cells with low basal Tensin4 expression. D. PLC and BEL7402 cells were treated with EGF at 40ng/ml for the indicated durations. The cell lysates were then collected and subjected for Western blotting. E. The Tensin4 mRNA expression level in BEL7402 cells after EGF induction at 6 and 24 hours were determined by qPCR comparing with the untreated control. F. SMMC-7721 cells subjected to EGF induction for the indicated time points were fixed and subjected to immunofluorescence detection for ERK1/2 and phospho-ERK1/2. The coverslips were counterstained with DAPI for nuclei. Scale bar: 10 μm.

    Journal: Oncotarget

    Article Title: Tensin4 is up-regulated by EGF-induced ERK1/2 activity and promotes cell proliferation and migration in hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: EGF-induced Tensin4 up-regulation required persistent ERK activation underlying ERK nuclear translocation A. SMMC-7721 cells were treated with EGF for the indicated durations and then released by washing twice with PBS followed by incubation for up to 8 hours. The cell lysates were then collected and subjected to Western blotting. B. SMMC-7721 and LO2 cells were induced with EGF treatment plus pre-treatment or post-treatment with the MEK inhibitor U0126 at the indicated time points. All the samples were collected at 360 minutes (6 hours) and subjected to Western blotting for the indicated proteins. C. PLC, SMMC-7721 and BEL7402 cells with different basal Tensin4 expression level were treated with EGF for 24 hours. The cell lysates were then collected and subjected to Western blotting. The arrowhead highlights the induction of Tensin4 in BEL7402 cells with low basal Tensin4 expression. D. PLC and BEL7402 cells were treated with EGF at 40ng/ml for the indicated durations. The cell lysates were then collected and subjected for Western blotting. E. The Tensin4 mRNA expression level in BEL7402 cells after EGF induction at 6 and 24 hours were determined by qPCR comparing with the untreated control. F. SMMC-7721 cells subjected to EGF induction for the indicated time points were fixed and subjected to immunofluorescence detection for ERK1/2 and phospho-ERK1/2. The coverslips were counterstained with DAPI for nuclei. Scale bar: 10 μm.

    Article Snippet: Epidermal growth factor (EGF) and inhibitor treatment 1.5×105 cells were seeded overnight in 12-well format and stimulated with 40ng/ml EGF (Cell Signaling Technology, Beverly, CA) in full growth medium for the indicated duration.

    Techniques: Activation Assay, Translocation Assay, Incubation, Western Blot, Planar Chromatography, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence