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  • 99
    Thermo Fisher epidermal growth factor egf
    N-WASP VCA is necessary for actin–microspike formation induced by N-WASP. ( A ) Confirmation of chimerization. Cell lysates of COS7 cells overexpressing wild-type N-WASP and N-WASP/WAVE1 VCA were subjected to Western blotting with anti-N-WASP VCA and anti-WAVE1 VCA antibodies. ( B ) Quantitation of microspike and membrane ruffle formation. <t>Transfected</t> cells were serum-starved and then stimulated with or without <t>EGF</t> for 10 min. The percentage of cells forming microspikes or membrane ruffles was calculated among transfected cells. Error bars represent the standard deviation of three different determinations. At least 50 cells were counted in each determination. ( C–E ) Immunofluorescence staining of transfected cells. Cells were stained with phalloidin to visualize actin filaments and antibody against N-WASP VCA or WAVE1 VCA to detect ectopically expressed proteins.
    Epidermal Growth Factor Egf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1897 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore egf stimulation
    scFvE3 is a selective sensor of RhoB activation in <t>HeLa</t> 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 <t>EGF</t> (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.
    Egf Stimulation, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore epidermal growth factor egf
    MKP-3 knockdown via siRNA increases ERK1/2 phosphorylation in H- ras MCF10A and DLD-1 cells. (A) Whole cell lysates were prepared from serum starved MCF10A and H- ras MCF10A cells. MKP-3 ( top panel ) was detected by immunoblot analysis. The blot was stripped and reprobed for β-tubulin ( bottom panel ) as a loading control. Whole cell lysates were prepared from (B) H- ras MCF10A cells or (C) DLD-1 cells that were either not transfected (lane 1), were transfected with duplex siRNA targeted against MKP-3 (lane 2), or were transfected with duplex scrambled siRNA (lane 3). The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. As a positive control for phospho-MEK1/2, nontransfected cells were incubated for 120 minutes with (B) 1.6 nM <t>TPA</t> (lane 4), or incubated for 30 minutes with (C) 3 nM <t>EGF</t> (lane 4). ). The graphs represent average pERK1/2 densitometry relative to non-transfected controls for (B) 10 independent experiments and (C) 6 independent experiments; error bars represent SEM. Filled bars (nt), not transfected. Open bars (M), transfected with duplex siRNA targeted against MKP-3. Hatched bars (S), transfected with duplex scrambled siRNA. Treatment of H- ras MCF10A and DLD-1 cells with MKP3 siRNA resulted in an increase in pERK1/2 levels relative to nontransfected control or treatment with scrambled siRNA (H- ras MCF10A: p = 0.006, n=30, 1-way ANOVA and Tukey’s HSD, α=0.05; DLD-1: p= 0.0008, n=18, 1-way ANOVA and Tukey’s HSD, α=0.05). We did not detect a difference between non transfected cells or cells transfected with scrambled siRNA.
    Epidermal Growth Factor Egf, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2527 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore egf
    <t>EGF</t> induces heparanase nucleolar localization in human BMBC cells A. and B. ). Human BMBC (231BR3) cells were serum starved for 16 hr at <t>37°C,</t> cell nucleoli were isolated as described in Materials and Methods, Nucleoli were analyzed following stimulation with EGF (0 – 150 ng/ml) for 30 min (A.), or EGF treatment for 0 – 120 min at 100 ng/ml (B.). C. and D. Human BMBC cells (231P and 231BR3) were not treated (control) or treated with EGF (100 ng/ml for 30 min following serum starvation for 16 hr). Next, immunofluorescent analyses were performed using confocal microscopy as described in Materials and Methods . Rabbit polyclonal HPSE antibody 1453 and mouse monoclonal antibody to HPSE were used as primary antibodies (Figures 2C and 2D respectively). They were diluted 1:1000 followed by secondary antibody Alexa Fluor 488 goat anti-rabbit IgG or anti-mouse IgG (green staining). Immunofluorescent staining was subsequently analyzed by confocal microscopy. Arrows indicate heparanase presence in nucleoli of 231BR3 cells. Red fluorescence indicates propidium iodide (P.I.) staining for cell nuclei. Images were taken at an original objective magnification of × 60. Representative images of four independent experiments are shown. E. Staining specificity for anti-HPSE polyclonal and monoclonal antibodies. Anti-rabbit (left panels) and anti-mouse (right panels) naive isotypic antibodies (IgG) were used as controls for staining specificity. Cell presence was determined by parallel P.I. staining. Immunofluorescence and confocal microscopy were subsequently performed, per above. Images were taken at an original objective magnification of × 60. F. Quantitation of HPSE nucleolar staining in 231P and 231BR3 cells treated or not treated with EGF (100 ng/ml for 30 min. at 37°C). Percentage of nucleolar HPSE staining was calculated from the number of nucleoli positive for HPSE staining compared to total nucleolar number obtained by fibrillarin staining. Bars represent measurements of ten fields for each treatment condition and from three independent experiments. ** P
    Egf, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cell Signaling Technology Inc epidermal growth factor egf
    Effect of <t>afatinib</t> on levels of phosphorylated forms of EGFR, AKT, and ERK in the HNE-1 cell line. Notes: Some cells were incubated with 10 ng/mL <t>EGF</t> for 30 minutes. Afatinib (5 μM) was administered as described in the “Materials and methods” section. Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular regulated protein kinases; FCS, fetal calf serum; GADPH, glyceraldehyde-3-phosphate dehydrogenase; HNE-1, human NPC cell line; NPC, nasopharyngeal carcinoma; p, phosphorylated.
    Epidermal Growth Factor Egf, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore epidermal growth factor egf stimuli
    Effect of <t>afatinib</t> on levels of phosphorylated forms of EGFR, AKT, and ERK in the HNE-1 cell line. Notes: Some cells were incubated with 10 ng/mL <t>EGF</t> for 30 minutes. Afatinib (5 μM) was administered as described in the “Materials and methods” section. Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular regulated protein kinases; FCS, fetal calf serum; GADPH, glyceraldehyde-3-phosphate dehydrogenase; HNE-1, human NPC cell line; NPC, nasopharyngeal carcinoma; p, phosphorylated.
    Epidermal Growth Factor Egf Stimuli, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    N-WASP VCA is necessary for actin–microspike formation induced by N-WASP. ( A ) Confirmation of chimerization. Cell lysates of COS7 cells overexpressing wild-type N-WASP and N-WASP/WAVE1 VCA were subjected to Western blotting with anti-N-WASP VCA and anti-WAVE1 VCA antibodies. ( B ) Quantitation of microspike and membrane ruffle formation. Transfected cells were serum-starved and then stimulated with or without EGF for 10 min. The percentage of cells forming microspikes or membrane ruffles was calculated among transfected cells. Error bars represent the standard deviation of three different determinations. At least 50 cells were counted in each determination. ( C–E ) Immunofluorescence staining of transfected cells. Cells were stained with phalloidin to visualize actin filaments and antibody against N-WASP VCA or WAVE1 VCA to detect ectopically expressed proteins.

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

    Article Title: Two tandem verprolin homology domains are necessary for a strong activation of Arp2/3 complex-induced actin polymerization and induction of microspike formation by N-WASP

    doi:

    Figure Lengend Snippet: N-WASP VCA is necessary for actin–microspike formation induced by N-WASP. ( A ) Confirmation of chimerization. Cell lysates of COS7 cells overexpressing wild-type N-WASP and N-WASP/WAVE1 VCA were subjected to Western blotting with anti-N-WASP VCA and anti-WAVE1 VCA antibodies. ( B ) Quantitation of microspike and membrane ruffle formation. Transfected cells were serum-starved and then stimulated with or without EGF for 10 min. The percentage of cells forming microspikes or membrane ruffles was calculated among transfected cells. Error bars represent the standard deviation of three different determinations. At least 50 cells were counted in each determination. ( C–E ) Immunofluorescence staining of transfected cells. Cells were stained with phalloidin to visualize actin filaments and antibody against N-WASP VCA or WAVE1 VCA to detect ectopically expressed proteins.

    Article Snippet: For the stimulation with epidermal growth factor (EGF), the transfected cells were starved for 15 h before treatment with 100 ng/ml human EGF (GIBCO/BRL) for 10 min.

    Techniques: Western Blot, Quantitation Assay, Transfection, Standard Deviation, Immunofluorescence, Staining

    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

    NHE1 co-localizes with ERK2 shown by immunofluorescence. a Immunofluorescence images of AP-1 cells (hNHE1 WT, D3-AXA and F2-AA) treated or not for 15 min with EGF (100 ng/ml). Merged images were zoomed to highlight the co-localization of ERK1/2 and NHE1 ( white arrowheads ). All other variants are shown in Additional file 6 : Figure S6. Data are representative of three independent biological replicates. b Representative line scans across membrane areas of images as in a . Line scans were performed using Olympus image analysis software. The figure shows the pixel intensities at each wavelength over the line shown, in the absence and presence of EGF as shown. Data are examples based on analysis of at least 60 cells in three to four independent replicates per condition. c Summary of line scan analysis data for all variants. The figure shows the percentage of cells with NHE1-ERK1/2 co-localization in both membranes, based on the experiments illustrated in b . Data are shown as mean percentage with SEM error bars, based on analysis of at least 60 cells in three to five independent replicates per condition. §§ and §§§, p

    Journal: BMC Biology

    Article Title: The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    doi: 10.1186/s12915-016-0252-7

    Figure Lengend Snippet: NHE1 co-localizes with ERK2 shown by immunofluorescence. a Immunofluorescence images of AP-1 cells (hNHE1 WT, D3-AXA and F2-AA) treated or not for 15 min with EGF (100 ng/ml). Merged images were zoomed to highlight the co-localization of ERK1/2 and NHE1 ( white arrowheads ). All other variants are shown in Additional file 6 : Figure S6. Data are representative of three independent biological replicates. b Representative line scans across membrane areas of images as in a . Line scans were performed using Olympus image analysis software. The figure shows the pixel intensities at each wavelength over the line shown, in the absence and presence of EGF as shown. Data are examples based on analysis of at least 60 cells in three to four independent replicates per condition. c Summary of line scan analysis data for all variants. The figure shows the percentage of cells with NHE1-ERK1/2 co-localization in both membranes, based on the experiments illustrated in b . Data are shown as mean percentage with SEM error bars, based on analysis of at least 60 cells in three to five independent replicates per condition. §§ and §§§, p

    Article Snippet: EGF-mediated stimulation of ERK1/2 activity in AP-1 cells Untransfected AP-1 cells or AP-1 cells expressing WT or variant hNHE1 were grown to ~ 80 % confluence in 10 cm Petri dishes, and incubated for 15 min in absence or presence of 100 ng/ml recombinant human EGF (Sigma).

    Techniques: Immunofluorescence, Software

    NHE1 regulates the phosphorylation status of cellular ERK1/2. ERK1/2 phosphorylation in untransfected AP-1 cells or AP-1 cells expressing WT and variant hNHE1, as indicated. Cells were stimulated or not with human recombinant EGF (100 ng/ml) for 15 min. a Representative immunoblots of p-ERK1/2 (T202/Y204 (ERK1)-T185/Y187 (ERK2) phosphorylation) and total ERK1/2 under the conditions shown. The arrows indicate ERK1 ( top ) and ERK2 ( bottom ), respectively. b Summary data from three to eight independent biological replicates per condition. Blots were scanned, and band intensities quantified using Un-Scan-IT Graph Digitizer software (Silk Scientific), as described in the “ Methods ”. Quantified data are shown as means with SEM error bars, normalized to those in untransfected, unstimulated AP-1 cells. *, **, and ***, p

    Journal: BMC Biology

    Article Title: The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    doi: 10.1186/s12915-016-0252-7

    Figure Lengend Snippet: NHE1 regulates the phosphorylation status of cellular ERK1/2. ERK1/2 phosphorylation in untransfected AP-1 cells or AP-1 cells expressing WT and variant hNHE1, as indicated. Cells were stimulated or not with human recombinant EGF (100 ng/ml) for 15 min. a Representative immunoblots of p-ERK1/2 (T202/Y204 (ERK1)-T185/Y187 (ERK2) phosphorylation) and total ERK1/2 under the conditions shown. The arrows indicate ERK1 ( top ) and ERK2 ( bottom ), respectively. b Summary data from three to eight independent biological replicates per condition. Blots were scanned, and band intensities quantified using Un-Scan-IT Graph Digitizer software (Silk Scientific), as described in the “ Methods ”. Quantified data are shown as means with SEM error bars, normalized to those in untransfected, unstimulated AP-1 cells. *, **, and ***, p

    Article Snippet: EGF-mediated stimulation of ERK1/2 activity in AP-1 cells Untransfected AP-1 cells or AP-1 cells expressing WT or variant hNHE1 were grown to ~ 80 % confluence in 10 cm Petri dishes, and incubated for 15 min in absence or presence of 100 ng/ml recombinant human EGF (Sigma).

    Techniques: Expressing, Variant Assay, Recombinant, Western Blot, Software

    Active AKT induces the up- and downregulation of MKRN1 and PTEN. ( a,b ) EGF-dependent AKT activation stabilizes MKRN1 and destabilizes PTEN. ME-180 cells were serum starved (0.2% serum) for 24 h, treated with 100 ng ml −1 EGF for the indicated time and then analysed by immunoblotting (right panel). Total RNA was purified from EGF (100 ng ml −1 )-stimulated ME-180 cells, and mRNA levels of MKRN1 and PTEN were measured using real-time PCR (left panel; data shown are mean±s.d.; n =3). ( c ) The effect of EGF treatment on the up- and downregulation of MKRN1 and PTEN, respectively, is PI3K/AKT dependent. After serum starvation, ME-180 cells were treated with EGF (100 ng ml −1 ) and LY294002 (PI3K inhibitor) or MK-2206 (1 μM, AKT inhibitor), as indicated. ( d ) EGF-dependent destabilization of PTEN is pAKT/MKRN1 axis dependent. ME-180 cells were transfected with AKT1 siRNA (siAKT1) or siMKRN1 #7, serum starved for 24 h after transfection and subsequently treated with EGF (100 ng ml −1 ) at the indicated time point. Cells were harvested at 72 h post transfection and analysed by immunoblotting. ( e,f ) The constitutively active PI3K mutants up- and downregulate MKRN1 and PTEN, respectively. Immunoblotting of lysates from ME-180 cells stably expressing PIK3CA p110α E545K or H1047R ( e ). ME-180 cells stably expressing PIK3CA p110α E545K were transfected with siMKRN1 #5 or siMKRN1 #7 and analysed by immunoblotting ( f ). ( a,c,e ) Relative PTEN protein expression levels are reported below the corresponding western blot bands.

    Journal: Nature Communications

    Article Title: PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis

    doi: 10.1038/ncomms8769

    Figure Lengend Snippet: Active AKT induces the up- and downregulation of MKRN1 and PTEN. ( a,b ) EGF-dependent AKT activation stabilizes MKRN1 and destabilizes PTEN. ME-180 cells were serum starved (0.2% serum) for 24 h, treated with 100 ng ml −1 EGF for the indicated time and then analysed by immunoblotting (right panel). Total RNA was purified from EGF (100 ng ml −1 )-stimulated ME-180 cells, and mRNA levels of MKRN1 and PTEN were measured using real-time PCR (left panel; data shown are mean±s.d.; n =3). ( c ) The effect of EGF treatment on the up- and downregulation of MKRN1 and PTEN, respectively, is PI3K/AKT dependent. After serum starvation, ME-180 cells were treated with EGF (100 ng ml −1 ) and LY294002 (PI3K inhibitor) or MK-2206 (1 μM, AKT inhibitor), as indicated. ( d ) EGF-dependent destabilization of PTEN is pAKT/MKRN1 axis dependent. ME-180 cells were transfected with AKT1 siRNA (siAKT1) or siMKRN1 #7, serum starved for 24 h after transfection and subsequently treated with EGF (100 ng ml −1 ) at the indicated time point. Cells were harvested at 72 h post transfection and analysed by immunoblotting. ( e,f ) The constitutively active PI3K mutants up- and downregulate MKRN1 and PTEN, respectively. Immunoblotting of lysates from ME-180 cells stably expressing PIK3CA p110α E545K or H1047R ( e ). ME-180 cells stably expressing PIK3CA p110α E545K were transfected with siMKRN1 #5 or siMKRN1 #7 and analysed by immunoblotting ( f ). ( a,c,e ) Relative PTEN protein expression levels are reported below the corresponding western blot bands.

    Article Snippet: In vivo and in vitro phosphorylation assays To detect ectopically expressed or endogenous phosphorylated MKRN1, EGF-stimulated cells were lysed in lysis buffer containing a 1:100 dilution of phosphatase inhibitor cocktail (p5726, Sigma-Aldrich).

    Techniques: Activation Assay, Purification, Real-time Polymerase Chain Reaction, Transfection, Stable Transfection, Expressing, Western Blot

    Active AKT induces the stabilization of MKRN1. ( a ) Overexpression of HA-tagged Myr-AKT but not K179M induces increased levels of endogenous MKRN1 or ectopically expressed FLAG-MKRN1 in H1299 cells. ( b ) Myr-AKT stabilizes endogenous MKRN1. H1299 cells were transfected with the indicated plasmid for 24 h and then treated with CHX (100 μg ml −1 ) at the indicated time points. ( c ) The half-life of the endogenous MKRN1 protein was determined in EGF (100 ng ml −1 )-stimulated ME-180 cells. ( b,c ) The amount of MKRN1 was determined using western blotting after normalization to actin. (bottom panel, data shown are means±s.d.; n =3). ( d ) H1299 cells transfected with the indicated plasmid were treated with 10 μM MG132 or LLnL for 4 h. ( e ) EGF-dependent MKRN1 ubiquitination is reduced by AKT ablation. ME-180 cells transduced with siAKT1 were treated with EGF (100 ng ml −1 ), followed by MG132 (10 μM) for 4 h. The lysates were immunoprecipitated using an anti-MKRN1 antibody, followed by immunoblotting with an HRP-conjugated anti-Ub antibody under denaturing conditions. ( f ) The protein half-life of the S109D mutant is longer than that of the WT protein. H1299 cells were transfected with FLAG-MKRN1 WT or S109D and then treated with CHX (100 μg ml −1 ) for the indicated time points. Bottom panel: the graphs indicate the relative amounts of MKRN1 protein compared with the levels of actin in the western blot (data shown are means±s.d.; n =3). ( g ) Ubiquitination status of the S109D mutant. H1299 cells were transfected with the indicated plasmids and then treated with MG132 (10 μM). Cells were lysed in 6 M guanidine-HCl, and ubiquitinated proteins were purified using Ni 2+ -NTA beads. His-purified proteins were detected by immunoblotting.

    Journal: Nature Communications

    Article Title: PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis

    doi: 10.1038/ncomms8769

    Figure Lengend Snippet: Active AKT induces the stabilization of MKRN1. ( a ) Overexpression of HA-tagged Myr-AKT but not K179M induces increased levels of endogenous MKRN1 or ectopically expressed FLAG-MKRN1 in H1299 cells. ( b ) Myr-AKT stabilizes endogenous MKRN1. H1299 cells were transfected with the indicated plasmid for 24 h and then treated with CHX (100 μg ml −1 ) at the indicated time points. ( c ) The half-life of the endogenous MKRN1 protein was determined in EGF (100 ng ml −1 )-stimulated ME-180 cells. ( b,c ) The amount of MKRN1 was determined using western blotting after normalization to actin. (bottom panel, data shown are means±s.d.; n =3). ( d ) H1299 cells transfected with the indicated plasmid were treated with 10 μM MG132 or LLnL for 4 h. ( e ) EGF-dependent MKRN1 ubiquitination is reduced by AKT ablation. ME-180 cells transduced with siAKT1 were treated with EGF (100 ng ml −1 ), followed by MG132 (10 μM) for 4 h. The lysates were immunoprecipitated using an anti-MKRN1 antibody, followed by immunoblotting with an HRP-conjugated anti-Ub antibody under denaturing conditions. ( f ) The protein half-life of the S109D mutant is longer than that of the WT protein. H1299 cells were transfected with FLAG-MKRN1 WT or S109D and then treated with CHX (100 μg ml −1 ) for the indicated time points. Bottom panel: the graphs indicate the relative amounts of MKRN1 protein compared with the levels of actin in the western blot (data shown are means±s.d.; n =3). ( g ) Ubiquitination status of the S109D mutant. H1299 cells were transfected with the indicated plasmids and then treated with MG132 (10 μM). Cells were lysed in 6 M guanidine-HCl, and ubiquitinated proteins were purified using Ni 2+ -NTA beads. His-purified proteins were detected by immunoblotting.

    Article Snippet: In vivo and in vitro phosphorylation assays To detect ectopically expressed or endogenous phosphorylated MKRN1, EGF-stimulated cells were lysed in lysis buffer containing a 1:100 dilution of phosphatase inhibitor cocktail (p5726, Sigma-Aldrich).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Transduction, Immunoprecipitation, Mutagenesis, Purification

    AKT phosphorylates serine 109 of MKRN1. ( a,b ) The interaction between ectopically expressed MKRN1 and AKT1 was demonstrated using a co-immunoprecipitation assay. ( c ) A GST pull-down assay revealed the direct interaction between MKRN1 and AKT1. GST-MKRN1 purified from bacteria and in vitro translated HA-AKT1 were incubated under cell-free conditions, and GST-MKRN1 was pulled down by glutathione Sepharose beads. ( d ) Endogenous MKRN1 was immunoprecipitated from ME-180 cells with or without EGF treatment (100 ng ml −1 ), and MKRN1-bound endogenous AKT1 was immunoblotted. ( e ) A consensus AKT phosphorylation site is present in MKRN1. ( f ) AKT directly phosphorylates MKRN1 WT but not the S109A mutant. An in vitro phosphorylation assay was performed using bacterially produced GST-AKT1 and FLAG-MKRN1 (WT or S109A), which was purified from FLAG-MKRN1-transfected HEK293T cells. Purified proteins were incubated with [γ- 32 P]ATP, and 32 P incorporation was detected by autoradiography. ( g ) Constitutively active, but not inactive, AKT phosphorylates MKRN1 on serine 109. H1299 cells were co-transfected with FLAG-MKRN1 (WT or S109A) and HA-tagged Myr-AKT or K179M. Phosphorylation of ectopically expressed MKRN1 was detected by an in vivo phosphorylation assay (IP panel); WCL was also analysed. ( h ) AKT phosphorylates MKRN1 upon EGF treatment. After serum starvation, ME-180 cells were stimulated by EGF (100 ng ml −1 ) in the absence or presence of MG132 (10 μM) for 4 h and were examined in an in vivo phosphorylation assay. Endogenous phospho-MKRN1 was immunoprecipitated using an anti-phosphoserine antibody (IP panel), and WCL was immunoblotted. ( i ) EGF-induced MKRN1 phosphorylation is inhibited by AKT ablation. ME-180 cells were transduced with control siRNA or AKT1 siRNA (siAKT1) and subsequently serum starved, followed by treatment with EGF (100 ng ml −1 ). Cell extracts were analysed using an in vivo phosphorylation assay. Endogenous phospho-MKRN1 is indicated in the IP panel.

    Journal: Nature Communications

    Article Title: PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis

    doi: 10.1038/ncomms8769

    Figure Lengend Snippet: AKT phosphorylates serine 109 of MKRN1. ( a,b ) The interaction between ectopically expressed MKRN1 and AKT1 was demonstrated using a co-immunoprecipitation assay. ( c ) A GST pull-down assay revealed the direct interaction between MKRN1 and AKT1. GST-MKRN1 purified from bacteria and in vitro translated HA-AKT1 were incubated under cell-free conditions, and GST-MKRN1 was pulled down by glutathione Sepharose beads. ( d ) Endogenous MKRN1 was immunoprecipitated from ME-180 cells with or without EGF treatment (100 ng ml −1 ), and MKRN1-bound endogenous AKT1 was immunoblotted. ( e ) A consensus AKT phosphorylation site is present in MKRN1. ( f ) AKT directly phosphorylates MKRN1 WT but not the S109A mutant. An in vitro phosphorylation assay was performed using bacterially produced GST-AKT1 and FLAG-MKRN1 (WT or S109A), which was purified from FLAG-MKRN1-transfected HEK293T cells. Purified proteins were incubated with [γ- 32 P]ATP, and 32 P incorporation was detected by autoradiography. ( g ) Constitutively active, but not inactive, AKT phosphorylates MKRN1 on serine 109. H1299 cells were co-transfected with FLAG-MKRN1 (WT or S109A) and HA-tagged Myr-AKT or K179M. Phosphorylation of ectopically expressed MKRN1 was detected by an in vivo phosphorylation assay (IP panel); WCL was also analysed. ( h ) AKT phosphorylates MKRN1 upon EGF treatment. After serum starvation, ME-180 cells were stimulated by EGF (100 ng ml −1 ) in the absence or presence of MG132 (10 μM) for 4 h and were examined in an in vivo phosphorylation assay. Endogenous phospho-MKRN1 was immunoprecipitated using an anti-phosphoserine antibody (IP panel), and WCL was immunoblotted. ( i ) EGF-induced MKRN1 phosphorylation is inhibited by AKT ablation. ME-180 cells were transduced with control siRNA or AKT1 siRNA (siAKT1) and subsequently serum starved, followed by treatment with EGF (100 ng ml −1 ). Cell extracts were analysed using an in vivo phosphorylation assay. Endogenous phospho-MKRN1 is indicated in the IP panel.

    Article Snippet: In vivo and in vitro phosphorylation assays To detect ectopically expressed or endogenous phosphorylated MKRN1, EGF-stimulated cells were lysed in lysis buffer containing a 1:100 dilution of phosphatase inhibitor cocktail (p5726, Sigma-Aldrich).

    Techniques: Co-Immunoprecipitation Assay, Pull Down Assay, Purification, In Vitro, Incubation, Immunoprecipitation, Mutagenesis, Phosphorylation Assay, Produced, Transfection, Autoradiography, In Vivo, Transduction

    EGF induces PTEN ubiquitination in an AKT/MKRN1-dependent manner. ( a ) Endogenous PTEN ubiquitination is accelerated upon EGF treatment. ME-180 cells treated with EGF for the indicated times were lysed and immunoblotted with an anti-PTEN antibody in 1% SDS buffer, followed by immunoblotting with an HRP-conjugated anti-Ub antibody. ( b ) EGF-induced PTEN ubiquitination is blocked by AKT ablation. ME-180 cells were transduced with control siRNA or siAKT1 and then treated with EGF (100 ng ml −1 ) for 24 h. Ubiquitinated PTEN was immunoprecipitated and immunoblotted as described above. ( c ) EGF-induced PTEN ubiquitination was blocked by MKRN1 ablation. ME-180 cells transduced with siMKRN1 #7 were stimulated by EGF (100 ng ml −1 ) for 24 h. To validate endogenous PTEN ubiquitination, an in vivo ubiquitination assay was performed as described above. ( d ) Knockdown of NEDD4-1 or WWP2 in ME-180 cells. Immunoblotting of NEDD4-1 or WWP2 and PTEN in four types of NEDD4-1 siRNA- or WWP2 siRNA-transfected cells. ( e ) MKRN1, but not NEDD4-1, or WWP2 ablation suppresses EGF-dependent PTEN ubiquitination. ME-180 cells were transfected with control siRNA, siMKRN1 #7, NEDD4-1 #3 or WWP2 #5 as indicated and subsequently stimulated with EGF for 24 h. Endogenous PTEN ubiquitination was analysed by an in vivo ubiquitination assay.

    Journal: Nature Communications

    Article Title: PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis

    doi: 10.1038/ncomms8769

    Figure Lengend Snippet: EGF induces PTEN ubiquitination in an AKT/MKRN1-dependent manner. ( a ) Endogenous PTEN ubiquitination is accelerated upon EGF treatment. ME-180 cells treated with EGF for the indicated times were lysed and immunoblotted with an anti-PTEN antibody in 1% SDS buffer, followed by immunoblotting with an HRP-conjugated anti-Ub antibody. ( b ) EGF-induced PTEN ubiquitination is blocked by AKT ablation. ME-180 cells were transduced with control siRNA or siAKT1 and then treated with EGF (100 ng ml −1 ) for 24 h. Ubiquitinated PTEN was immunoprecipitated and immunoblotted as described above. ( c ) EGF-induced PTEN ubiquitination was blocked by MKRN1 ablation. ME-180 cells transduced with siMKRN1 #7 were stimulated by EGF (100 ng ml −1 ) for 24 h. To validate endogenous PTEN ubiquitination, an in vivo ubiquitination assay was performed as described above. ( d ) Knockdown of NEDD4-1 or WWP2 in ME-180 cells. Immunoblotting of NEDD4-1 or WWP2 and PTEN in four types of NEDD4-1 siRNA- or WWP2 siRNA-transfected cells. ( e ) MKRN1, but not NEDD4-1, or WWP2 ablation suppresses EGF-dependent PTEN ubiquitination. ME-180 cells were transfected with control siRNA, siMKRN1 #7, NEDD4-1 #3 or WWP2 #5 as indicated and subsequently stimulated with EGF for 24 h. Endogenous PTEN ubiquitination was analysed by an in vivo ubiquitination assay.

    Article Snippet: In vivo and in vitro phosphorylation assays To detect ectopically expressed or endogenous phosphorylated MKRN1, EGF-stimulated cells were lysed in lysis buffer containing a 1:100 dilution of phosphatase inhibitor cocktail (p5726, Sigma-Aldrich).

    Techniques: Transduction, Immunoprecipitation, In Vivo, Ubiquitin Assay, Transfection

    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

    (A) Responses of MCF-7 cells co-expressing redCALWY-4 and ER-eZinCh-2 to the addition of 10 ng mL –1 EGF together with 500 nM ionomycin. (B) Responses of TamR cells co-expressing redCALWY-4 and ER-eCALWY-4 to the addition of 10 ng mL –1 EGF together with 500 nM ionomycin. Traces in A and B represent the average of three cells after normalization at t = 0 s. Error bars represent SEM.

    Journal: Metallomics

    Article Title: Monitoring cytosolic and ER Zn2+ in stimulated breast cancer cells using genetically encoded FRET sensors in stimulated breast cancer cells using genetically encoded FRET sensors †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5mt00257eClick here for additional data file.

    doi: 10.1039/c5mt00257e

    Figure Lengend Snippet: (A) Responses of MCF-7 cells co-expressing redCALWY-4 and ER-eZinCh-2 to the addition of 10 ng mL –1 EGF together with 500 nM ionomycin. (B) Responses of TamR cells co-expressing redCALWY-4 and ER-eCALWY-4 to the addition of 10 ng mL –1 EGF together with 500 nM ionomycin. Traces in A and B represent the average of three cells after normalization at t = 0 s. Error bars represent SEM.

    Article Snippet: For EGF/ionomycin stimulation, cells were perfused with HEPES buffer without additives for a few minutes, followed by perfusion with HEPES buffer containing 10 ng mL–1 EGF (Sigma, E9644) and 500 nM ionomycin (Sigma, 56092-82-1).

    Techniques: Expressing

    Responses of MCF-7 cells expressing cytosolic eCALWY-4 (A) and TamR cells expressing cytosolic eZinCh-2 (B) to the addition of 10 ng mL –1 EGF together with 500 nM of the calcium ionophore ionomycin. Responses of MCF-7 cells expressing ER-eZinCh-2 (C) and TamR cells expressing ER-eCALWY-4 (D) to the addition of 10 ng mL –1 EGF together with 500 nM of the calcium ionophore ionomycin. All traces represent the average of three cells after normalization at t = 0 s. Error bars represent SEM.

    Journal: Metallomics

    Article Title: Monitoring cytosolic and ER Zn2+ in stimulated breast cancer cells using genetically encoded FRET sensors in stimulated breast cancer cells using genetically encoded FRET sensors †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5mt00257eClick here for additional data file.

    doi: 10.1039/c5mt00257e

    Figure Lengend Snippet: Responses of MCF-7 cells expressing cytosolic eCALWY-4 (A) and TamR cells expressing cytosolic eZinCh-2 (B) to the addition of 10 ng mL –1 EGF together with 500 nM of the calcium ionophore ionomycin. Responses of MCF-7 cells expressing ER-eZinCh-2 (C) and TamR cells expressing ER-eCALWY-4 (D) to the addition of 10 ng mL –1 EGF together with 500 nM of the calcium ionophore ionomycin. All traces represent the average of three cells after normalization at t = 0 s. Error bars represent SEM.

    Article Snippet: For EGF/ionomycin stimulation, cells were perfused with HEPES buffer without additives for a few minutes, followed by perfusion with HEPES buffer containing 10 ng mL–1 EGF (Sigma, E9644) and 500 nM ionomycin (Sigma, 56092-82-1).

    Techniques: Expressing

    PI3KC2β increases MAPK and Akt signalling downstream of EGFR and PDGFR. (A) and (B) NIH3T3-V, -C2β-DN and -C2β-WT cells were serum-deprived overnight and stimulated for 10 min. with EGF (A) or PDGF (B) as indicated. Cell lysates were analysed by immunobloting and MAPK and Akt pathway activation was assessed with indicated phospho-specific antibodies. (C) and (D) Lysates of NIH3T3-V, -C2β-DN and -C2β-WT cells grown in 10% FCS were analysed for activated signalling molecules implicated in the cell cycle control and caveolae formation such as p53, Bcl2, PTEN and caveolin 1, −2. WT1, −2 and DN1, −2 indicate individual clones.

    Journal: PLoS ONE

    Article Title: Phosphoinositide 3-Kinase C2? Regulates RhoA and the Actin Cytoskeleton through an Interaction with Dbl

    doi: 10.1371/journal.pone.0044945

    Figure Lengend Snippet: PI3KC2β increases MAPK and Akt signalling downstream of EGFR and PDGFR. (A) and (B) NIH3T3-V, -C2β-DN and -C2β-WT cells were serum-deprived overnight and stimulated for 10 min. with EGF (A) or PDGF (B) as indicated. Cell lysates were analysed by immunobloting and MAPK and Akt pathway activation was assessed with indicated phospho-specific antibodies. (C) and (D) Lysates of NIH3T3-V, -C2β-DN and -C2β-WT cells grown in 10% FCS were analysed for activated signalling molecules implicated in the cell cycle control and caveolae formation such as p53, Bcl2, PTEN and caveolin 1, −2. WT1, −2 and DN1, −2 indicate individual clones.

    Article Snippet: Recombinant EGF, PDGF BB were purchased from Calbiochem, La Jolla,CA,USA.

    Techniques: Western Blot, Activation Assay, Clone Assay

    The assembly of the Dbl - PI3KC2β complex is not modulated by PI3K activity or cell stimulation with EGF or PDGF. (A) HEK293 cells were transfected with vectors encoding Dbl in combination with Myc-PI3KC2β WT or DN, or empty vector. Immunoprecipitates prepared with anti-Dbl or anti-Myc tag antibodies were analysed by western blot with the antibodies indicated. (B) Lysates from NIH3T3-V, -C2β-WT or -C2β-DN cells were immunoprecipitated with anti-Dbl antibodies and analysed by western blot. (C) Serum-starved NIH3T3-C2β-WT cells were stimulated with EGF (20 ng/ml) or PDGF (20 ng/ml) for the indicated lengths of time. Immunoprecipitates prepared with anti-Glu (EE) tag antibodies were analysed by western blot with the antibodies indicated. (D) HEK293 cells were transfected with vectors encoding Dbl in combination with Myc-PI3KC2β WT or DN, or empty vector. Immunoprecipitates prepared with anti-Dbl antibodies were analysed for GEF activity towards recombinant RhoA.

    Journal: PLoS ONE

    Article Title: Phosphoinositide 3-Kinase C2? Regulates RhoA and the Actin Cytoskeleton through an Interaction with Dbl

    doi: 10.1371/journal.pone.0044945

    Figure Lengend Snippet: The assembly of the Dbl - PI3KC2β complex is not modulated by PI3K activity or cell stimulation with EGF or PDGF. (A) HEK293 cells were transfected with vectors encoding Dbl in combination with Myc-PI3KC2β WT or DN, or empty vector. Immunoprecipitates prepared with anti-Dbl or anti-Myc tag antibodies were analysed by western blot with the antibodies indicated. (B) Lysates from NIH3T3-V, -C2β-WT or -C2β-DN cells were immunoprecipitated with anti-Dbl antibodies and analysed by western blot. (C) Serum-starved NIH3T3-C2β-WT cells were stimulated with EGF (20 ng/ml) or PDGF (20 ng/ml) for the indicated lengths of time. Immunoprecipitates prepared with anti-Glu (EE) tag antibodies were analysed by western blot with the antibodies indicated. (D) HEK293 cells were transfected with vectors encoding Dbl in combination with Myc-PI3KC2β WT or DN, or empty vector. Immunoprecipitates prepared with anti-Dbl antibodies were analysed for GEF activity towards recombinant RhoA.

    Article Snippet: Recombinant EGF, PDGF BB were purchased from Calbiochem, La Jolla,CA,USA.

    Techniques: Activity Assay, Cell Stimulation, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Recombinant

    MKP-3 knockdown via siRNA increases ERK1/2 phosphorylation in H- ras MCF10A and DLD-1 cells. (A) Whole cell lysates were prepared from serum starved MCF10A and H- ras MCF10A cells. MKP-3 ( top panel ) was detected by immunoblot analysis. The blot was stripped and reprobed for β-tubulin ( bottom panel ) as a loading control. Whole cell lysates were prepared from (B) H- ras MCF10A cells or (C) DLD-1 cells that were either not transfected (lane 1), were transfected with duplex siRNA targeted against MKP-3 (lane 2), or were transfected with duplex scrambled siRNA (lane 3). The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. As a positive control for phospho-MEK1/2, nontransfected cells were incubated for 120 minutes with (B) 1.6 nM TPA (lane 4), or incubated for 30 minutes with (C) 3 nM EGF (lane 4). ). The graphs represent average pERK1/2 densitometry relative to non-transfected controls for (B) 10 independent experiments and (C) 6 independent experiments; error bars represent SEM. Filled bars (nt), not transfected. Open bars (M), transfected with duplex siRNA targeted against MKP-3. Hatched bars (S), transfected with duplex scrambled siRNA. Treatment of H- ras MCF10A and DLD-1 cells with MKP3 siRNA resulted in an increase in pERK1/2 levels relative to nontransfected control or treatment with scrambled siRNA (H- ras MCF10A: p = 0.006, n=30, 1-way ANOVA and Tukey’s HSD, α=0.05; DLD-1: p= 0.0008, n=18, 1-way ANOVA and Tukey’s HSD, α=0.05). We did not detect a difference between non transfected cells or cells transfected with scrambled siRNA.

    Journal: Toxicology and applied pharmacology

    Article Title: Reciprocal Regulation of Extracellular Signal Regulated Kinase 1/2 and Mitogen Activated Protein Kinase Phosphatase-3

    doi: 10.1016/j.taap.2008.08.007

    Figure Lengend Snippet: MKP-3 knockdown via siRNA increases ERK1/2 phosphorylation in H- ras MCF10A and DLD-1 cells. (A) Whole cell lysates were prepared from serum starved MCF10A and H- ras MCF10A cells. MKP-3 ( top panel ) was detected by immunoblot analysis. The blot was stripped and reprobed for β-tubulin ( bottom panel ) as a loading control. Whole cell lysates were prepared from (B) H- ras MCF10A cells or (C) DLD-1 cells that were either not transfected (lane 1), were transfected with duplex siRNA targeted against MKP-3 (lane 2), or were transfected with duplex scrambled siRNA (lane 3). The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. As a positive control for phospho-MEK1/2, nontransfected cells were incubated for 120 minutes with (B) 1.6 nM TPA (lane 4), or incubated for 30 minutes with (C) 3 nM EGF (lane 4). ). The graphs represent average pERK1/2 densitometry relative to non-transfected controls for (B) 10 independent experiments and (C) 6 independent experiments; error bars represent SEM. Filled bars (nt), not transfected. Open bars (M), transfected with duplex siRNA targeted against MKP-3. Hatched bars (S), transfected with duplex scrambled siRNA. Treatment of H- ras MCF10A and DLD-1 cells with MKP3 siRNA resulted in an increase in pERK1/2 levels relative to nontransfected control or treatment with scrambled siRNA (H- ras MCF10A: p = 0.006, n=30, 1-way ANOVA and Tukey’s HSD, α=0.05; DLD-1: p= 0.0008, n=18, 1-way ANOVA and Tukey’s HSD, α=0.05). We did not detect a difference between non transfected cells or cells transfected with scrambled siRNA.

    Article Snippet: 12- 0 -tetradecanoylphorbol-13-acetate (TPA), cholera toxin, hydrocortisone, insulin, cycloheximide, actinomycin D, and epidermal growth factor (EGF) were purchased from Sigma (St. Louis, MO).

    Techniques: Transfection, Positive Control, Incubation

    TPA and EGF stimulate the loss and recovery of MKP-3 protein. Whole cell lysates were prepared from (A) H- ras MCF10A cells that were incubated for the indicated times with 1.6 nM TPA; (B) H- ras MCF10A cells; or (C) DLD-1 cells that were incubated for the indicated times with 3 nM EGF. The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. The graphs represent average densitometry of pMEK1/2, pERK1/2, and MKP-3 for 3 independent experiments; error bars represent SEM. Filled bars, pMEK1/2. Open bars, pERK1/2. Hatched bars, MKP-3.

    Journal: Toxicology and applied pharmacology

    Article Title: Reciprocal Regulation of Extracellular Signal Regulated Kinase 1/2 and Mitogen Activated Protein Kinase Phosphatase-3

    doi: 10.1016/j.taap.2008.08.007

    Figure Lengend Snippet: TPA and EGF stimulate the loss and recovery of MKP-3 protein. Whole cell lysates were prepared from (A) H- ras MCF10A cells that were incubated for the indicated times with 1.6 nM TPA; (B) H- ras MCF10A cells; or (C) DLD-1 cells that were incubated for the indicated times with 3 nM EGF. The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. The graphs represent average densitometry of pMEK1/2, pERK1/2, and MKP-3 for 3 independent experiments; error bars represent SEM. Filled bars, pMEK1/2. Open bars, pERK1/2. Hatched bars, MKP-3.

    Article Snippet: 12- 0 -tetradecanoylphorbol-13-acetate (TPA), cholera toxin, hydrocortisone, insulin, cycloheximide, actinomycin D, and epidermal growth factor (EGF) were purchased from Sigma (St. Louis, MO).

    Techniques: Incubation

    Hrs and Tsg101 are not required for lysosomal degradation of PAR1. (A) PAR1-expressing HeLa cells transiently transfected with nonspecific (ns), Hrs-, or Tsg101-specific siRNAs were incubated in the absence or presence of 100 μM SFLLRN for 90 min at 37°C, and the amount of PAR1 protein degradation was then assessed as described above. Cell lysates were immunoblotted with anti-Hrs or -Tsg101 antibodies to confirm loss of these proteins in siRNA-transfected cells or for actin expression to control for equal loading. The results (mean ± SE) shown in the graph are represented as a percentage of PAR1 remaining compared with untreated control cells for each transfection condition and are an average of at least three experiments. (B) PAR1-expressing HeLa cells were treated with or without 2 mM leupeptin and then incubated with 100 μM SFLLRN as described above. Cells were fixed and coimmunostained for PAR1 (green) and LAMP1 (red) and imaged by confocal microscopy. Colocalization of PAR1 and LAMP1 is revealed by the yellow color in the merged image (arrowheads). The insets are magnifications of boxed areas. Scale bar, 10 μm. (C) Serum-starved HeLa cells transfected with Tsg101, Hrs, or nonspecific (ns) siRNAs were incubated in the absence or presence of 100 ng/ml EGF ligand for 30 min at 37°C, and the amount of receptor protein remaining was then determined by immunoblot analysis using anti-EGFR antibodies. Cell lysates run in parallel were immunoblotted for Hrs and Tsg101 to confirm loss of protein in siRNA-transfected cells or for actin expression to control for equal loading. Similar results were observed in three separate experiments

    Journal: Molecular Biology of the Cell

    Article Title: An Essential Role for SNX1 in Lysosomal Sorting of Protease-activated Receptor-1: Evidence for Retromer-, Hrs-, and Tsg101-independent Functions of Sorting Nexins

    doi: 10.1091/mbc.E05-09-0899

    Figure Lengend Snippet: Hrs and Tsg101 are not required for lysosomal degradation of PAR1. (A) PAR1-expressing HeLa cells transiently transfected with nonspecific (ns), Hrs-, or Tsg101-specific siRNAs were incubated in the absence or presence of 100 μM SFLLRN for 90 min at 37°C, and the amount of PAR1 protein degradation was then assessed as described above. Cell lysates were immunoblotted with anti-Hrs or -Tsg101 antibodies to confirm loss of these proteins in siRNA-transfected cells or for actin expression to control for equal loading. The results (mean ± SE) shown in the graph are represented as a percentage of PAR1 remaining compared with untreated control cells for each transfection condition and are an average of at least three experiments. (B) PAR1-expressing HeLa cells were treated with or without 2 mM leupeptin and then incubated with 100 μM SFLLRN as described above. Cells were fixed and coimmunostained for PAR1 (green) and LAMP1 (red) and imaged by confocal microscopy. Colocalization of PAR1 and LAMP1 is revealed by the yellow color in the merged image (arrowheads). The insets are magnifications of boxed areas. Scale bar, 10 μm. (C) Serum-starved HeLa cells transfected with Tsg101, Hrs, or nonspecific (ns) siRNAs were incubated in the absence or presence of 100 ng/ml EGF ligand for 30 min at 37°C, and the amount of receptor protein remaining was then determined by immunoblot analysis using anti-EGFR antibodies. Cell lysates run in parallel were immunoblotted for Hrs and Tsg101 to confirm loss of protein in siRNA-transfected cells or for actin expression to control for equal loading. Similar results were observed in three separate experiments

    Article Snippet: The epidermal growth factor (EGF) ligand and leupeptin were purchased from Sigma (St. Louis, MO).

    Techniques: Expressing, Transfection, Incubation, Confocal Microscopy

    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

    Effects of E‐cadherin‐Fc chimera protein (E‐cad‐Fc) on epithelial‐mesenchymal transition ( EMT ) and cancer stem cell markers in SW 480 colon cancer cells. SW 480 cells were divided into three groups, control group, E‐cad‐Fc(+) group, and epithelial growth factor ( EGF ) + transforming growth factor ( TGF )β1(+) group. A, Time course study of cell morphology (24, 48, and 72 h). Cells were cultured on normal tissue culture plate (left), cultured on a non‐treated plate coated with E‐cad‐Fc matrix (middle), or cultured on normal tissue culture plate with medium supplemented by EGF and TGF ‐β1 (right). Mesenchymal‐like cells that showed a spindle‐like shape appeared as time passed in the E‐cad‐Fc(+) and EGF + TGF ‐β1(+) groups. B, Expressions of epithelial or mesenchymal markers at the protein level when treated for 48 h with E‐cad‐Fc matrix or EGF and TGF ‐β1. Quantitative analysis in three independent experiments is shown in the right panel. N‐cadherin expression was significantly upregulated in the E‐cad‐Fc–treated group (* P

    Journal: Cancer Science

    Article Title: E‐cadherin‐Fc chimera protein matrix enhances cancer stem‐like properties and induces mesenchymal features in colon cancer cells, et al. E‐cadherin‐Fc chimera protein matrix enhances cancer stem‐like properties and induces mesenchymal features in colon cancer cells

    doi: 10.1111/cas.14193

    Figure Lengend Snippet: Effects of E‐cadherin‐Fc chimera protein (E‐cad‐Fc) on epithelial‐mesenchymal transition ( EMT ) and cancer stem cell markers in SW 480 colon cancer cells. SW 480 cells were divided into three groups, control group, E‐cad‐Fc(+) group, and epithelial growth factor ( EGF ) + transforming growth factor ( TGF )β1(+) group. A, Time course study of cell morphology (24, 48, and 72 h). Cells were cultured on normal tissue culture plate (left), cultured on a non‐treated plate coated with E‐cad‐Fc matrix (middle), or cultured on normal tissue culture plate with medium supplemented by EGF and TGF ‐β1 (right). Mesenchymal‐like cells that showed a spindle‐like shape appeared as time passed in the E‐cad‐Fc(+) and EGF + TGF ‐β1(+) groups. B, Expressions of epithelial or mesenchymal markers at the protein level when treated for 48 h with E‐cad‐Fc matrix or EGF and TGF ‐β1. Quantitative analysis in three independent experiments is shown in the right panel. N‐cadherin expression was significantly upregulated in the E‐cad‐Fc–treated group (* P

    Article Snippet: Briefly, after cells were seeded, they were treated with TGF‐β1 (2.5 ng/mL; Sigma‐Aldrich) and epithelial growth factor (EGF) (10 ng/mL; Sigma‐Aldrich) for 48 hours.

    Techniques: Cell Culture, Expressing

    EGF induces heparanase nucleolar localization in human BMBC cells A. and B. ). Human BMBC (231BR3) cells were serum starved for 16 hr at 37°C, cell nucleoli were isolated as described in Materials and Methods, Nucleoli were analyzed following stimulation with EGF (0 – 150 ng/ml) for 30 min (A.), or EGF treatment for 0 – 120 min at 100 ng/ml (B.). C. and D. Human BMBC cells (231P and 231BR3) were not treated (control) or treated with EGF (100 ng/ml for 30 min following serum starvation for 16 hr). Next, immunofluorescent analyses were performed using confocal microscopy as described in Materials and Methods . Rabbit polyclonal HPSE antibody 1453 and mouse monoclonal antibody to HPSE were used as primary antibodies (Figures 2C and 2D respectively). They were diluted 1:1000 followed by secondary antibody Alexa Fluor 488 goat anti-rabbit IgG or anti-mouse IgG (green staining). Immunofluorescent staining was subsequently analyzed by confocal microscopy. Arrows indicate heparanase presence in nucleoli of 231BR3 cells. Red fluorescence indicates propidium iodide (P.I.) staining for cell nuclei. Images were taken at an original objective magnification of × 60. Representative images of four independent experiments are shown. E. Staining specificity for anti-HPSE polyclonal and monoclonal antibodies. Anti-rabbit (left panels) and anti-mouse (right panels) naive isotypic antibodies (IgG) were used as controls for staining specificity. Cell presence was determined by parallel P.I. staining. Immunofluorescence and confocal microscopy were subsequently performed, per above. Images were taken at an original objective magnification of × 60. F. Quantitation of HPSE nucleolar staining in 231P and 231BR3 cells treated or not treated with EGF (100 ng/ml for 30 min. at 37°C). Percentage of nucleolar HPSE staining was calculated from the number of nucleoli positive for HPSE staining compared to total nucleolar number obtained by fibrillarin staining. Bars represent measurements of ten fields for each treatment condition and from three independent experiments. ** P

    Journal: Molecular cancer research : MCR

    Article Title: EGF-induced Heparanase Nucleolar Localization Augments DNA Topoisomerase I Activity in Brain Metastatic Breast Cancer

    doi: 10.1158/1541-7786.MCR-09-0375

    Figure Lengend Snippet: EGF induces heparanase nucleolar localization in human BMBC cells A. and B. ). Human BMBC (231BR3) cells were serum starved for 16 hr at 37°C, cell nucleoli were isolated as described in Materials and Methods, Nucleoli were analyzed following stimulation with EGF (0 – 150 ng/ml) for 30 min (A.), or EGF treatment for 0 – 120 min at 100 ng/ml (B.). C. and D. Human BMBC cells (231P and 231BR3) were not treated (control) or treated with EGF (100 ng/ml for 30 min following serum starvation for 16 hr). Next, immunofluorescent analyses were performed using confocal microscopy as described in Materials and Methods . Rabbit polyclonal HPSE antibody 1453 and mouse monoclonal antibody to HPSE were used as primary antibodies (Figures 2C and 2D respectively). They were diluted 1:1000 followed by secondary antibody Alexa Fluor 488 goat anti-rabbit IgG or anti-mouse IgG (green staining). Immunofluorescent staining was subsequently analyzed by confocal microscopy. Arrows indicate heparanase presence in nucleoli of 231BR3 cells. Red fluorescence indicates propidium iodide (P.I.) staining for cell nuclei. Images were taken at an original objective magnification of × 60. Representative images of four independent experiments are shown. E. Staining specificity for anti-HPSE polyclonal and monoclonal antibodies. Anti-rabbit (left panels) and anti-mouse (right panels) naive isotypic antibodies (IgG) were used as controls for staining specificity. Cell presence was determined by parallel P.I. staining. Immunofluorescence and confocal microscopy were subsequently performed, per above. Images were taken at an original objective magnification of × 60. F. Quantitation of HPSE nucleolar staining in 231P and 231BR3 cells treated or not treated with EGF (100 ng/ml for 30 min. at 37°C). Percentage of nucleolar HPSE staining was calculated from the number of nucleoli positive for HPSE staining compared to total nucleolar number obtained by fibrillarin staining. Bars represent measurements of ten fields for each treatment condition and from three independent experiments. ** P

    Article Snippet: Cells were serum-starved overnight (16 hrs) at 37°C, then stimulated with EGF (E) (Sigma) or serum-free medium only (S) as indicated in figures legends before being lysed using lysis buffer (Cell Signaling Technology, Danvers, MA).

    Techniques: Isolation, Confocal Microscopy, Staining, Fluorescence, Immunofluorescence, Quantitation Assay

    Effects of recombinant human HPSE on Topo I activity in 231BR3 nucleoli Representative experiments where recombinant human active HPSE (rhaHPSE) ( A. ) and inactive HPSE (rhiHPSE) ( B. ) were added to nucleoli isolated from 231BR3 cells (no EGF treatment) at indicated concentrations (0, 10, and 100 ng/ml), following incubation for 30 min at 37°C with supercoiled DNA (100 ng) provided by the TopoGen kit. Topo I activity was examined by 1% agarose gel electrophoresis with ethidium bromide and visualized using UV transilluminator, and determined by DNA status. Relaxed DNA (Rel) indicates Topo I activity while supercoiled DNA (Sc) indicates a lack of Topo I activity. Rel and Sc DNA were used as positive/negative marker controls, respectively. Lower panels were prepared upon densitometric analyses from three independent experiments. Bars represent means plus SD measurements from three independent experiments. * P

    Journal: Molecular cancer research : MCR

    Article Title: EGF-induced Heparanase Nucleolar Localization Augments DNA Topoisomerase I Activity in Brain Metastatic Breast Cancer

    doi: 10.1158/1541-7786.MCR-09-0375

    Figure Lengend Snippet: Effects of recombinant human HPSE on Topo I activity in 231BR3 nucleoli Representative experiments where recombinant human active HPSE (rhaHPSE) ( A. ) and inactive HPSE (rhiHPSE) ( B. ) were added to nucleoli isolated from 231BR3 cells (no EGF treatment) at indicated concentrations (0, 10, and 100 ng/ml), following incubation for 30 min at 37°C with supercoiled DNA (100 ng) provided by the TopoGen kit. Topo I activity was examined by 1% agarose gel electrophoresis with ethidium bromide and visualized using UV transilluminator, and determined by DNA status. Relaxed DNA (Rel) indicates Topo I activity while supercoiled DNA (Sc) indicates a lack of Topo I activity. Rel and Sc DNA were used as positive/negative marker controls, respectively. Lower panels were prepared upon densitometric analyses from three independent experiments. Bars represent means plus SD measurements from three independent experiments. * P

    Article Snippet: Cells were serum-starved overnight (16 hrs) at 37°C, then stimulated with EGF (E) (Sigma) or serum-free medium only (S) as indicated in figures legends before being lysed using lysis buffer (Cell Signaling Technology, Danvers, MA).

    Techniques: Recombinant, Activity Assay, Isolation, Incubation, Agarose Gel Electrophoresis, Marker

    Effect of afatinib on levels of phosphorylated forms of EGFR, AKT, and ERK in the HNE-1 cell line. Notes: Some cells were incubated with 10 ng/mL EGF for 30 minutes. Afatinib (5 μM) was administered as described in the “Materials and methods” section. Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular regulated protein kinases; FCS, fetal calf serum; GADPH, glyceraldehyde-3-phosphate dehydrogenase; HNE-1, human NPC cell line; NPC, nasopharyngeal carcinoma; p, phosphorylated.

    Journal: Drug Design, Development and Therapy

    Article Title: In vitro and in vivo efficacy of afatinib as a single agent or in combination with gemcitabine for the treatment of nasopharyngeal carcinoma

    doi: 10.2147/DDDT.S94432

    Figure Lengend Snippet: Effect of afatinib on levels of phosphorylated forms of EGFR, AKT, and ERK in the HNE-1 cell line. Notes: Some cells were incubated with 10 ng/mL EGF for 30 minutes. Afatinib (5 μM) was administered as described in the “Materials and methods” section. Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular regulated protein kinases; FCS, fetal calf serum; GADPH, glyceraldehyde-3-phosphate dehydrogenase; HNE-1, human NPC cell line; NPC, nasopharyngeal carcinoma; p, phosphorylated.

    Article Snippet: Western blotting Cells were treated with afatinib for 24 hours and then stimulated with 10 nM epidermal growth factor (EGF) (Cell Signaling Technology, Beverley, MA, USA) for 30 minutes before harvest.

    Techniques: Incubation

    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