epidermal growth factor egf Search Results


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  • 99
    Lonza human epidermal growth factor
    Human Epidermal Growth Factor, supplied by Lonza, used in various techniques. Bioz Stars score: 99/100, based on 387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher egf
    <t>EGF</t> and <t>HRGβ</t> stimulation of MDA-MB-435 cells induces recruitment of the p85 subunit of PI 3-K to the phosphotyrosine cellular fraction. Cells were serum starved for 24 h and harvested as previously described. Cells (7.5 × 10 6 per sample) were incubated in the absence or presence of 100 ng/ml EGF (A) or 100 ng/ml HRGβ (B) for the indicated periods at 37°C. Cells were then lysed, and phosphotyrosine-containing proteins were immunoprecipitated with the anti-phosphotyrosine mAb PY20 coupled to protein A-Sepharose beads. Washed immunocomplexes were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Western blotting was performed on membranes with the anti-phosphotyrosine mAb 4G10 to detect phosphotyrosine-containing proteins (upper panels). Blots were stripped and reprobed for EGFR or erbB3 (A and B, respectively; our unpublished data) and for p85 (lower panels). Similar data were obtained in at least two additional assays.
    Egf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore epidermal growth factor egf
    Overexpression of Myc and Gfi1b transforms cerebellar stem cells. (A) The CS and CSCG cells grow as spheres in the presence of <t>EGF</t> and <t>bFGF</t> growth factors. The red (tdT) and green (GFP) florescence show expression of Myc and Gfi1b proteins, respectively. Scale bar = 50 μm. CS: C erebellar stem cells expressing S endai virus c-protein; CSCG: C erebellar stem cells expressing S endai virus c-protein, Myc, and G fi1b. (B) CS cells expressing Myc, and Gfi1b proteins form tumors in the cerebellum of NSG immunodeficient mice. The resulting cell line prepared from NSG brain tumors, called CSCG, is highly tumorigenic in hCD46Tg mice. Tumor growth was monitored by bioluminescence imaging. (C) Western blots showing higher expression of Myc and Gfi1b in CSCG cells compared with the parental CS cells. The expression level of MycN, Gfi1, and p53 proteins were the same in both cell lines.
    Epidermal Growth Factor Egf, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher epidermal growth factor egf
    Proliferating HaCaT cells were more sensitive to EISO-induced plasma membrane damage than quiescent cells HaCaT cells were starved in serum-free DMEM for 24 hr. Fresh media supplemented with bovine pituitary extract and epidermal growth factor (A, C, E, G) or not supplemented (B, D, F, H) was added to the cells for 3 hr prior to treatment with EISO. At the indicated times, cells were detached by trypsinization and pooled with floating cells. Cells were pelleted, resuspended in PBS and incubated with fluorescein diacetate and propidium iodide for 15 minutes before analysis by flow cytometry. Using control cells loaded with individual dyes, regions of cells were identified as live cells with uncompromised membranes (R2), live cells with compromised membranes (R3), or severely compromised cells. Serum-starved EISO-treated cells (6 hr; panel D) displayed 2 distinct PI-stained populations of actively cycling cells in G 1  and G 2  phases (R7) that was used to confirm the population of cells with compromised membranes (evidenced by reduced fluorescein retention) but that were still alive.
    Epidermal Growth Factor Egf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 2023 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human recombinant epidermal growth factor
    Proliferating HaCaT cells were more sensitive to EISO-induced plasma membrane damage than quiescent cells HaCaT cells were starved in serum-free DMEM for 24 hr. Fresh media supplemented with bovine pituitary extract and epidermal growth factor (A, C, E, G) or not supplemented (B, D, F, H) was added to the cells for 3 hr prior to treatment with EISO. At the indicated times, cells were detached by trypsinization and pooled with floating cells. Cells were pelleted, resuspended in PBS and incubated with fluorescein diacetate and propidium iodide for 15 minutes before analysis by flow cytometry. Using control cells loaded with individual dyes, regions of cells were identified as live cells with uncompromised membranes (R2), live cells with compromised membranes (R3), or severely compromised cells. Serum-starved EISO-treated cells (6 hr; panel D) displayed 2 distinct PI-stained populations of actively cycling cells in G 1  and G 2  phases (R7) that was used to confirm the population of cells with compromised membranes (evidenced by reduced fluorescein retention) but that were still alive.
    Human Recombinant Epidermal Growth Factor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1496 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore egf
    Dual role of p38 in ligand-independent stress-triggered EGFR trafficking in HeLa cells. ( a ) EGFR immunolocalization in untreated versus UVC-exposed cells 1 h post UVC exposure (left), and quantification of surface downregulation in cells exposed to UVC or treated with <t>EGF</t> for the indicated times (right). Arrows indicate perinuclear accumulation of EGFR (green) after UVC exposure. Data are mean±s.e.m. of three independent experiments. ( b ) Untreated or UVC-exposed HeLa cells were fixed after 1 h, or further incubated with the p38 inhibitor <t>SB202190</t> (SB) for 1 h (left). Untreated or EGF-treated cells were fixed after 30 min, or further incubated with SB for 1 h in the continuous presence of EGF (right). p38 inhibition causes EGFR (green) redistribution to the plasma membrane following UVC, but not EGF exposure. ( c ) Pre-treatment for 30 min with PITSTOPII prevents UVC-induced EGFR (green) internalization (left) but PITSTOPII addition 1 h after UVC-induced EGFR internalization does not affect perinuclear EGFR accumulation or the recycling induced by simultaneous p38 inhibition (right). ( d ) Cells transfected with control or Rab11 siRNA were immunoblotted after 72 h for Rab11 and tubulin to assess knockdown efficiency (top). Rab11 knockdown did not prevent UVC-induced EGFR (green) internalization but prevented EGFR recycling after subsequent SB treatment (bottom). ( e ) Cells transfected with EGFR-GFP were fixed 1 h after UVC exposure and ultrathin cryosections were immuno-labelled for EGFR with 8 nm gold. EGFR-GFP (arrows) is on the limiting membrane and ILVs of MVBs. ( f ) Immunofluorescence analysis of UVC and EGF sequentially exposed HeLa cells (see Methods for experimental details). Red arrows show endosomes containing EGFR (green) and EGF (red). White arrows show EGFR+ve, EGF-ve endosomes, indicating a separate subset of MVBs containing stress-internalized but not EGF-bound EGFR. Scale bar, 5 μm. ( g ) Cells transfected with constitutively active Rab5-Q79L-DsRed were exposed to UVC and incubated for 1 h before treatment with EGF-AlexaFluor 647 (red) for 3 h. Red and white arrows show EGFR+ve/EGF+ve and EGFR+ve/EGF-ve endosomes, respectively. Scale bars, 10 μm for confocal and 100 nm for EM, unless otherwise indicated; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
    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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    Thermo Fisher recombinant egf
    Effect of <t>BDNF</t> on <t>EGF-induced</t> human NSPC proliferation and migration. Quantitative analysis of human NSPCs proliferation ( A ) and migration ( B ) were determined by BrdU incorporation and Transwell assay following 2 hours treatment of different concentrations of BDNF stimulation with 20 ng/mL EGF or not. (B) Data were expressed as mean ratio compared with the control group; ( C ) Cells incubated with EGF or EGF and BDNF. Nuclei were counter-stained with DAPI; ( D ) Analysis of apoptosis by Flow-cytometry. There was no appreciable change in these groups CON: control (incubated with medium without EGF or BDNF). * p
    Recombinant Egf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore human epidermal growth factor
    Effect of <t>BDNF</t> on <t>EGF-induced</t> human NSPC proliferation and migration. Quantitative analysis of human NSPCs proliferation ( A ) and migration ( B ) were determined by BrdU incorporation and Transwell assay following 2 hours treatment of different concentrations of BDNF stimulation with 20 ng/mL EGF or not. (B) Data were expressed as mean ratio compared with the control group; ( C ) Cells incubated with EGF or EGF and BDNF. Nuclei were counter-stained with DAPI; ( D ) Analysis of apoptosis by Flow-cytometry. There was no appreciable change in these groups CON: control (incubated with medium without EGF or BDNF). * p
    Human Epidermal Growth Factor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher recombinant human epidermal growth factor
    Rif activity was inhibited upon mitogenic stimulation mediated by MAPK or PI3K activation. ( A ) Domain structure of mDia1. Abbreviations: G, GTPase binding region necessary for RhoA binding; DID, diaphanous inhibitory domain; DD, dimerization domain; CC, coiled coil; FH1, formin homology 1 domain; FH2, formin homology 2 domain; DAD, diaphanous autoinhibitory domain; ( B ) pGEX-4T1-RifWT (1–195 aa) and pGEX-4T1-mDia1-G-DID (73–370 aa) <t>recombinant</t> proteins were generated in an E. coli protein expression system and soluble protein was immobilized to glutathione-Sepharose 4B beads for the use in a Rif activation assay. GST-Rif (1–195 aa) was cleaved by thrombin and then the released Rif was loaded with GDP or GTP. mDia1-G-DID beads were then incubated with GDP- or GTP-loaded Rif at 4 °C for 1 h. The beads were washed three times with lysis buffer and eluted with sample buffer for immunoblotting. HEK293 cell lysate was added to mimic the complex intracellular environment; ( C ) HeLa cells were serum starved overnight, then treated with 100 ng/mL <t>Epidermal</t> <t>Growth</t> <t>Factor</t> (EGF), 8 ng/mL Lysophosphatidic Acid (LPA), 1 µg/mL Sphingosine 1-Phosphate (SIP) or 10% serum for 3 min and Rif activation levels then examined by the Rif activation assay described in ( B ); ( D ) HeLa cells were serum starved overnight, then pretreated with or without p38 MAPK, PI3K, or p42/44 MAPK inhibitors for 30 min and then stimulated with 100 ng/mL EGF for 5 min. Rif activation levels were examined by the Rif activation assay. Rif activation levels were quantified by immunoblotting. Data are means ± S.E.M ( n = 3). * p
    Recombinant Human Epidermal Growth Factor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 481 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech recombinant human epidermal growth factor
    Rif activity was inhibited upon mitogenic stimulation mediated by MAPK or PI3K activation. ( A ) Domain structure of mDia1. Abbreviations: G, GTPase binding region necessary for RhoA binding; DID, diaphanous inhibitory domain; DD, dimerization domain; CC, coiled coil; FH1, formin homology 1 domain; FH2, formin homology 2 domain; DAD, diaphanous autoinhibitory domain; ( B ) pGEX-4T1-RifWT (1–195 aa) and pGEX-4T1-mDia1-G-DID (73–370 aa) <t>recombinant</t> proteins were generated in an E. coli protein expression system and soluble protein was immobilized to glutathione-Sepharose 4B beads for the use in a Rif activation assay. GST-Rif (1–195 aa) was cleaved by thrombin and then the released Rif was loaded with GDP or GTP. mDia1-G-DID beads were then incubated with GDP- or GTP-loaded Rif at 4 °C for 1 h. The beads were washed three times with lysis buffer and eluted with sample buffer for immunoblotting. HEK293 cell lysate was added to mimic the complex intracellular environment; ( C ) HeLa cells were serum starved overnight, then treated with 100 ng/mL <t>Epidermal</t> <t>Growth</t> <t>Factor</t> (EGF), 8 ng/mL Lysophosphatidic Acid (LPA), 1 µg/mL Sphingosine 1-Phosphate (SIP) or 10% serum for 3 min and Rif activation levels then examined by the Rif activation assay described in ( B ); ( D ) HeLa cells were serum starved overnight, then pretreated with or without p38 MAPK, PI3K, or p42/44 MAPK inhibitors for 30 min and then stimulated with 100 ng/mL EGF for 5 min. Rif activation levels were examined by the Rif activation assay. Rif activation levels were quantified by immunoblotting. Data are means ± S.E.M ( n = 3). * p
    Recombinant Human Epidermal Growth Factor, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson epidermal growth factor egf
    Transactivation of HER2 by <t>leptin</t> . MCF-7 cells were stimulated for 15 min with 100, 200, 500 ng/mL doses of leptin (Lep) or <t>EGF.</t> The expression of Tyr 1248 HER2 (p-HER2), total HER2 levels (HER2) was studied in 100 μg of total proteins by WB with specific Abs, as described in Materials and Methods. The levels of GAPDH in the same blots were assessed to control protein loading. The graph represents levels of Tyr 1248 HER2 relative to total HER2 under different stimuli. Bars represent SE.
    Epidermal Growth Factor Egf, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 289 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher egf recombinant human protein
    Characteristics of OSCs. (a) The representative images of SP cells from K7M2 and MG63 osteosarcoma cell lines. The median value of K7M2 and MG63 SP cells was 1.25% and 1.07%, respectively. n = 5. (b) The representative TEM images of autophagosomes in K7M2 and MG63 SP cells. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (c) Tumor spheres of K7M2 and MG63 osteosarcoma cells after culturing in the serum-free medium <t>DMEM/F12-bFGF-EGF-B27</t> for 7 days. The parental K7M2 and MG63 cells cultured in DMEM/F12 supplemented with 1% FBS served as a control. Scale bars = 50 μ m. n = 5. (d) Western blot analysis of the pluripotent transcription factors Sox2, Oct4, and Nanog and the autophagy markers LC3, ATG5, and ATG7 in K7M2 and MG63 OSCs. Data are shown as mean ± SD, n = 3. (e) The mRNA expression levels of the pluripotency-associated genes SOX2 , OCT4 , and NANOG and the autophagy-related genes ATG5 and ATG7 . n = 3. (f) Immunofluorescence analysis of autophagy in K7M2 and MG63 SP cells. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 200 μ m. n = 3. (g) Osteogenic and chondrogenic differentiation of K7M2 and MG63 SP cells. Cells differentiated into osteoblasts and chondroblasts were detected by staining with Alizarin Red and Alcian Blue. Scale bars = 100 μ m. n = 3. (h) Flow cytometry-based assay for the pluripotent transcription factors Sox2 and Oct4 and the CSC surface markers CD44, CD105, CD133, and Stro-1 in K7M2 and MG63 SP cells. n = 3. ∗ P
    Egf Recombinant Human Protein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 687 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human epidermal growth factor
    Adiponectin inhibits <t>growth</t> <t>factor-mediated</t> proliferation of prostatic epithelial and stromal cells. ( a , b ) CCK-8 proliferation analysis of RWPE1 and WPMY1 cells cultured in regular growth medium, with 0, 1, 5, 10 or 20 μg/ml <t>human</t> <t>recombinant</t> adiponectin treatment. QD 450 values were converted to cell numbers according to the standard curve. (one-way analysis of variance followed by Dunnett’s post-tests; n = 5; *p
    Recombinant Human Epidermal Growth Factor, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 345 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc egf
    <t>EGF</t> induced morphological changes in cells with Tensin4 up-regulation SMMC-7721, LO2, PLC and BEL7402 cells were treated with EGF at a concentration of <t>40ng/ml</t> for 24 hours. The cells were fixed and counterstained with phalloidin and DAPI for F-actin and cell nuclei. Scale bar: 10 μm.
    Egf, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech human recombinant epidermal growth factor
    <t>EGF</t> induced morphological changes in cells with Tensin4 up-regulation SMMC-7721, LO2, PLC and BEL7402 cells were treated with EGF at a concentration of <t>40ng/ml</t> for 24 hours. The cells were fixed and counterstained with phalloidin and DAPI for F-actin and cell nuclei. Scale bar: 10 μm.
    Human Recombinant Epidermal Growth Factor, supplied by PeproTech, used in various techniques. Bioz Stars score: 94/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher biotinylated egf
    Targeting QDs to EGFR. EGFR labeled with <t>biotinylated</t> <t>EGF</t> (bioEGF), followed by staining with aminoQDs covalently conjugated to streptavidin (not drawn to scale).
    Biotinylated Egf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore megf
    Targeting QDs to EGFR. EGFR labeled with <t>biotinylated</t> <t>EGF</t> (bioEGF), followed by staining with aminoQDs covalently conjugated to streptavidin (not drawn to scale).
    Megf, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore human epidermal growth factor egf
    Eupatolide inhibits the migration and invasion of breast cancer cells. (A) MDA-MB-231 and (B) MCF-7 cells were co-treated with either 100 ng/ml of <t>EGF</t> or 50 ng/ml of <t>TGF-β1,</t> as well as 10 µM eupatolide or a control, and the migration of cells was monitored for 24 h using an IncuCyte live-cell imaging system. After 24 h, relative wound density was acquired by IncuCyte. (C and D) MDA-MB-231 cells were seeded into Transwell chambers and stimulated with (C) 100 ng/ml of EGF or (D) 50 ng/ml TGF-β1 independently, or were co-treated with 10 µM eupatolide. The number of invaded cells in the bottom chamber was counted to measure invasion levels. *P
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    EGF and HRGβ stimulation of MDA-MB-435 cells induces recruitment of the p85 subunit of PI 3-K to the phosphotyrosine cellular fraction. Cells were serum starved for 24 h and harvested as previously described. Cells (7.5 × 10 6 per sample) were incubated in the absence or presence of 100 ng/ml EGF (A) or 100 ng/ml HRGβ (B) for the indicated periods at 37°C. Cells were then lysed, and phosphotyrosine-containing proteins were immunoprecipitated with the anti-phosphotyrosine mAb PY20 coupled to protein A-Sepharose beads. Washed immunocomplexes were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Western blotting was performed on membranes with the anti-phosphotyrosine mAb 4G10 to detect phosphotyrosine-containing proteins (upper panels). Blots were stripped and reprobed for EGFR or erbB3 (A and B, respectively; our unpublished data) and for p85 (lower panels). Similar data were obtained in at least two additional assays.

    Journal: Molecular Biology of the Cell

    Article Title: Stimulation of ?1-Integrin Function by Epidermal Growth Factor and Heregulin-? Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

    doi:

    Figure Lengend Snippet: EGF and HRGβ stimulation of MDA-MB-435 cells induces recruitment of the p85 subunit of PI 3-K to the phosphotyrosine cellular fraction. Cells were serum starved for 24 h and harvested as previously described. Cells (7.5 × 10 6 per sample) were incubated in the absence or presence of 100 ng/ml EGF (A) or 100 ng/ml HRGβ (B) for the indicated periods at 37°C. Cells were then lysed, and phosphotyrosine-containing proteins were immunoprecipitated with the anti-phosphotyrosine mAb PY20 coupled to protein A-Sepharose beads. Washed immunocomplexes were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Western blotting was performed on membranes with the anti-phosphotyrosine mAb 4G10 to detect phosphotyrosine-containing proteins (upper panels). Blots were stripped and reprobed for EGFR or erbB3 (A and B, respectively; our unpublished data) and for p85 (lower panels). Similar data were obtained in at least two additional assays.

    Article Snippet: Growth factor stimulation was performed with EGF (Life Technologies), betacellulin, HRGα, or HRGβ (all from R & D Systems, Minneapolis, MN).

    Techniques: Multiple Displacement Amplification, Incubation, Immunoprecipitation, SDS Page, Western Blot

    Overexpression of the wild-type or dominant negative p85 subunit of PI 3-K inhibits EGF- or HRGβ-mediated increases in MDA-MB-435 adhesion to COLL. Control vector expressing GFP (top panel) or constructs expressing a GFP-wt p85 (middle panel) or GFP-Δp85 (bottom panel) fusion protein were transiently transfected into MDA-MB-435 cells as described in MATERIALS AND METHODS. Transfected cells were allowed to recover for 24 h and then placed in serum-free media overnight. Cells were harvested as for standard adhesion assays, except that no Calcein AM labeling was performed, and cells (∼300,000 cells per well) were added to 24-well plates coated with 6 μg/well COLL. Adhesion was analyzed in the presence of PBS alone (open circles) or containing 1 μg/well TS2/16 (solid circles), 100 ng/ml EGF (solid diamonds), or 100 ng/ml HRGβ (solid squares) for 10 min at 37°C. Nonadherent cells were washed away, and adherent cells were collected fromwells using a 1:1 trypsin:1 mM EDTA solution. Collected cells were then analyzed by flow cytometry using aliquots of preadherent cell populations to confirm cell numbers added to wells for each transfectant and to determine the percent expression of GFP in the starting cell populations. Percent adhesion was determined by gating GFP-negative (−), GFP-low (+), -middle- (++), and -high (+++)-positive cells and comparing preadherent and adherent cell numbers from each population. The data shown reflect fold differences in adhesion when compared with the GFP-negative, unstimulated cell subpopulation from each transfectant. Average percent adhesion was determined from samples examined in triplicate for each stimulation condition, and results were similar for a minimum of three independent assays.

    Journal: Molecular Biology of the Cell

    Article Title: Stimulation of ?1-Integrin Function by Epidermal Growth Factor and Heregulin-? Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

    doi:

    Figure Lengend Snippet: Overexpression of the wild-type or dominant negative p85 subunit of PI 3-K inhibits EGF- or HRGβ-mediated increases in MDA-MB-435 adhesion to COLL. Control vector expressing GFP (top panel) or constructs expressing a GFP-wt p85 (middle panel) or GFP-Δp85 (bottom panel) fusion protein were transiently transfected into MDA-MB-435 cells as described in MATERIALS AND METHODS. Transfected cells were allowed to recover for 24 h and then placed in serum-free media overnight. Cells were harvested as for standard adhesion assays, except that no Calcein AM labeling was performed, and cells (∼300,000 cells per well) were added to 24-well plates coated with 6 μg/well COLL. Adhesion was analyzed in the presence of PBS alone (open circles) or containing 1 μg/well TS2/16 (solid circles), 100 ng/ml EGF (solid diamonds), or 100 ng/ml HRGβ (solid squares) for 10 min at 37°C. Nonadherent cells were washed away, and adherent cells were collected fromwells using a 1:1 trypsin:1 mM EDTA solution. Collected cells were then analyzed by flow cytometry using aliquots of preadherent cell populations to confirm cell numbers added to wells for each transfectant and to determine the percent expression of GFP in the starting cell populations. Percent adhesion was determined by gating GFP-negative (−), GFP-low (+), -middle- (++), and -high (+++)-positive cells and comparing preadherent and adherent cell numbers from each population. The data shown reflect fold differences in adhesion when compared with the GFP-negative, unstimulated cell subpopulation from each transfectant. Average percent adhesion was determined from samples examined in triplicate for each stimulation condition, and results were similar for a minimum of three independent assays.

    Article Snippet: Growth factor stimulation was performed with EGF (Life Technologies), betacellulin, HRGα, or HRGβ (all from R & D Systems, Minneapolis, MN).

    Techniques: Over Expression, Dominant Negative Mutation, Multiple Displacement Amplification, Plasmid Preparation, Expressing, Construct, Transfection, Labeling, Flow Cytometry, Cytometry

    Tyrosine phosphorylation of erbB2 after EGF or HRGβ stimulation is inhibited by the anti-erbB2 blocking Ab-16. MDA-MB-435 cells (5 × 10 6 per sample) were coated for 15 min on ice with either control mouse IgG (− lanes) or erbB2 Ab-16 (+ lanes) at 0.5 μg/1 × 10 6 cells. Cells were then stimulated for various times with 100 ng/ml EGF or HRGβ at 37°C followed by lysis in an equal volume of 2× lysis buffer. Immunoprecipitation for erbB2 was performed on cleared lysates, and samples were separated by SDS-PAGE. Immunoblotting for phosphotyrosine using 4G10 (upper panel) was performed after Western transfer. The membrane was then stripped and reprobed for erbB2 (lower panel) to confirm receptor levels. The figure shown is representative of a minimum of three independent assays.

    Journal: Molecular Biology of the Cell

    Article Title: Stimulation of ?1-Integrin Function by Epidermal Growth Factor and Heregulin-? Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

    doi:

    Figure Lengend Snippet: Tyrosine phosphorylation of erbB2 after EGF or HRGβ stimulation is inhibited by the anti-erbB2 blocking Ab-16. MDA-MB-435 cells (5 × 10 6 per sample) were coated for 15 min on ice with either control mouse IgG (− lanes) or erbB2 Ab-16 (+ lanes) at 0.5 μg/1 × 10 6 cells. Cells were then stimulated for various times with 100 ng/ml EGF or HRGβ at 37°C followed by lysis in an equal volume of 2× lysis buffer. Immunoprecipitation for erbB2 was performed on cleared lysates, and samples were separated by SDS-PAGE. Immunoblotting for phosphotyrosine using 4G10 (upper panel) was performed after Western transfer. The membrane was then stripped and reprobed for erbB2 (lower panel) to confirm receptor levels. The figure shown is representative of a minimum of three independent assays.

    Article Snippet: Growth factor stimulation was performed with EGF (Life Technologies), betacellulin, HRGα, or HRGβ (all from R & D Systems, Minneapolis, MN).

    Techniques: Blocking Assay, Multiple Displacement Amplification, Lysis, Immunoprecipitation, SDS Page, Western Blot

    Role of PI 3-K in EGF- and HRGβ-induced up-regulation of β1-Integrin function

    Journal: Molecular Biology of the Cell

    Article Title: Stimulation of ?1-Integrin Function by Epidermal Growth Factor and Heregulin-? Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

    doi:

    Figure Lengend Snippet: Role of PI 3-K in EGF- and HRGβ-induced up-regulation of β1-Integrin function

    Article Snippet: Growth factor stimulation was performed with EGF (Life Technologies), betacellulin, HRGα, or HRGβ (all from R & D Systems, Minneapolis, MN).

    Techniques:

    MDA-MB-435 cell adhesion is stimulated by multiple growth factors that bind to and activate members of the EGF receptor family. For analysis of dose response to growth factor, increasing amounts of EGF ranging from 1 pg/ml to 500 ng/ml were added to COLL-coated wells before addition of cells and stimulation at 37°C (A; solid diamonds). Adhesion to BSA-coated wells (open squares) was performed as a control. Dose responses for adhesion to COLL were determined in the presence of increasing amounts of HRGβ (B), the EGF-like growth factor betacellulin (C), or HRGα (D). Unstimulated (PBS; B–D, cross-hatched bars), EGF-stimulated (B–D, hatched bars), or TS2/16-stimulated (B–D, solid bars) adhesion was analyzed for comparison. Cells plated on BSA alone as a control for nonspecific adhesion generally showed

    Journal: Molecular Biology of the Cell

    Article Title: Stimulation of ?1-Integrin Function by Epidermal Growth Factor and Heregulin-? Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

    doi:

    Figure Lengend Snippet: MDA-MB-435 cell adhesion is stimulated by multiple growth factors that bind to and activate members of the EGF receptor family. For analysis of dose response to growth factor, increasing amounts of EGF ranging from 1 pg/ml to 500 ng/ml were added to COLL-coated wells before addition of cells and stimulation at 37°C (A; solid diamonds). Adhesion to BSA-coated wells (open squares) was performed as a control. Dose responses for adhesion to COLL were determined in the presence of increasing amounts of HRGβ (B), the EGF-like growth factor betacellulin (C), or HRGα (D). Unstimulated (PBS; B–D, cross-hatched bars), EGF-stimulated (B–D, hatched bars), or TS2/16-stimulated (B–D, solid bars) adhesion was analyzed for comparison. Cells plated on BSA alone as a control for nonspecific adhesion generally showed

    Article Snippet: Growth factor stimulation was performed with EGF (Life Technologies), betacellulin, HRGα, or HRGβ (all from R & D Systems, Minneapolis, MN).

    Techniques: Multiple Displacement Amplification

    Multiple growth factors induce tyrosine phosphorylation of the EGFR, erbB2, and erbB3 in MDA-MB-435 cells. Receptor activation was assessed after stimulation of MDA-MB-435 cells (10 × 10 6 cells per sample) for increasing periods at 37°C with PBS alone (first lane in each panel), EGF at 100 ng/ml, betacellulin (βCELL) at 10 ng/ml, HRGα at 100 ng/ml, or HRGβ at 100 ng/ml. Lysis buffer (2×) was added to each sample after the indicated stimulation time, and cleared lysates were immunoprecipitated for EGFR (A), erbB2 (B), or erbB3 (C). Western blotting was performed on samples after separation by SDS-PAGE and transfer to polyvinylidene difluoride membranes. Total phosphotyrosine content in each sample was assessed by probing with anti-phosphotyrosine Ab 4G10 (upper panels). Equivalent receptor loading was confirmed by stripping and reprobing each blot for the presence of the indicated receptor (lower panels). Blots were also stripped and reprobed for the presence of p85 (our unpublished data). Similar results were observed in at least three independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: Stimulation of ?1-Integrin Function by Epidermal Growth Factor and Heregulin-? Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

    doi:

    Figure Lengend Snippet: Multiple growth factors induce tyrosine phosphorylation of the EGFR, erbB2, and erbB3 in MDA-MB-435 cells. Receptor activation was assessed after stimulation of MDA-MB-435 cells (10 × 10 6 cells per sample) for increasing periods at 37°C with PBS alone (first lane in each panel), EGF at 100 ng/ml, betacellulin (βCELL) at 10 ng/ml, HRGα at 100 ng/ml, or HRGβ at 100 ng/ml. Lysis buffer (2×) was added to each sample after the indicated stimulation time, and cleared lysates were immunoprecipitated for EGFR (A), erbB2 (B), or erbB3 (C). Western blotting was performed on samples after separation by SDS-PAGE and transfer to polyvinylidene difluoride membranes. Total phosphotyrosine content in each sample was assessed by probing with anti-phosphotyrosine Ab 4G10 (upper panels). Equivalent receptor loading was confirmed by stripping and reprobing each blot for the presence of the indicated receptor (lower panels). Blots were also stripped and reprobed for the presence of p85 (our unpublished data). Similar results were observed in at least three independent experiments.

    Article Snippet: Growth factor stimulation was performed with EGF (Life Technologies), betacellulin, HRGα, or HRGβ (all from R & D Systems, Minneapolis, MN).

    Techniques: Multiple Displacement Amplification, Activation Assay, Lysis, Immunoprecipitation, Western Blot, SDS Page, Stripping Membranes

    Betacelluin, EGF, and HRGβ induce β1-integrin-dependent MDA-MB-435 cell migration on LAM. (A) Cells were grown to ∼75% confluency and placed in serum-free media for 16 h before harvesting as for adhesion assays. Polycarbonate filters (8 μm) were coated overnight at 4°C in PBS containing mouse EHS-LAM or EHS-COLL (our unpublished data) at 20 μg/ml. Forty-eight-well chemotaxis chambers were assembled with assay media alone or containing increasing amounts of growth factors in the lower chambers, as indicated. LAM-coated filters were placed over the lower wells and, ∼23,000 MDA-MB-435 cells were placed in the upper wells and allowed to migrate at 37°C for 4–6 h. Migrated cells in each well were quantitated on fixed and stained filters. The sum of four microscopic fields was taken for each well, and three wells were averaged for each stimulation condition. (B) Antibody blocking studies were carried out by preincubating cells with control IgG (open bars) or the inhibitory β1-integrin-specific mAb P5D2 (solid bars) at 1 μg/1 × 10 6 cells on ice for 10 min before adding the cells to the upper wells of chambers containing EGF or HRGβ and containing filters coated with LAM or COLL. Some variability was observed with the levels of basal migration in the data shown in A, because each growth factor titration was carried out in a separate chemotaxis chamber. However, the level of stimulation over that of basal migration for each growth factor was reproducible over at least three separate assays.

    Journal: Molecular Biology of the Cell

    Article Title: Stimulation of ?1-Integrin Function by Epidermal Growth Factor and Heregulin-? Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

    doi:

    Figure Lengend Snippet: Betacelluin, EGF, and HRGβ induce β1-integrin-dependent MDA-MB-435 cell migration on LAM. (A) Cells were grown to ∼75% confluency and placed in serum-free media for 16 h before harvesting as for adhesion assays. Polycarbonate filters (8 μm) were coated overnight at 4°C in PBS containing mouse EHS-LAM or EHS-COLL (our unpublished data) at 20 μg/ml. Forty-eight-well chemotaxis chambers were assembled with assay media alone or containing increasing amounts of growth factors in the lower chambers, as indicated. LAM-coated filters were placed over the lower wells and, ∼23,000 MDA-MB-435 cells were placed in the upper wells and allowed to migrate at 37°C for 4–6 h. Migrated cells in each well were quantitated on fixed and stained filters. The sum of four microscopic fields was taken for each well, and three wells were averaged for each stimulation condition. (B) Antibody blocking studies were carried out by preincubating cells with control IgG (open bars) or the inhibitory β1-integrin-specific mAb P5D2 (solid bars) at 1 μg/1 × 10 6 cells on ice for 10 min before adding the cells to the upper wells of chambers containing EGF or HRGβ and containing filters coated with LAM or COLL. Some variability was observed with the levels of basal migration in the data shown in A, because each growth factor titration was carried out in a separate chemotaxis chamber. However, the level of stimulation over that of basal migration for each growth factor was reproducible over at least three separate assays.

    Article Snippet: Growth factor stimulation was performed with EGF (Life Technologies), betacellulin, HRGα, or HRGβ (all from R & D Systems, Minneapolis, MN).

    Techniques: Multiple Displacement Amplification, Migration, Laser Capture Microdissection, Chemotaxis Assay, Staining, Blocking Assay, Titration

    Overexpression of Myc and Gfi1b transforms cerebellar stem cells. (A) The CS and CSCG cells grow as spheres in the presence of EGF and bFGF growth factors. The red (tdT) and green (GFP) florescence show expression of Myc and Gfi1b proteins, respectively. Scale bar = 50 μm. CS: C erebellar stem cells expressing S endai virus c-protein; CSCG: C erebellar stem cells expressing S endai virus c-protein, Myc, and G fi1b. (B) CS cells expressing Myc, and Gfi1b proteins form tumors in the cerebellum of NSG immunodeficient mice. The resulting cell line prepared from NSG brain tumors, called CSCG, is highly tumorigenic in hCD46Tg mice. Tumor growth was monitored by bioluminescence imaging. (C) Western blots showing higher expression of Myc and Gfi1b in CSCG cells compared with the parental CS cells. The expression level of MycN, Gfi1, and p53 proteins were the same in both cell lines.

    Journal: Neuro-Oncology

    Article Title: An oncolytic measles virus–sensitive Group 3 medulloblastoma model in immune-competent mice

    doi: 10.1093/neuonc/noy089

    Figure Lengend Snippet: Overexpression of Myc and Gfi1b transforms cerebellar stem cells. (A) The CS and CSCG cells grow as spheres in the presence of EGF and bFGF growth factors. The red (tdT) and green (GFP) florescence show expression of Myc and Gfi1b proteins, respectively. Scale bar = 50 μm. CS: C erebellar stem cells expressing S endai virus c-protein; CSCG: C erebellar stem cells expressing S endai virus c-protein, Myc, and G fi1b. (B) CS cells expressing Myc, and Gfi1b proteins form tumors in the cerebellum of NSG immunodeficient mice. The resulting cell line prepared from NSG brain tumors, called CSCG, is highly tumorigenic in hCD46Tg mice. Tumor growth was monitored by bioluminescence imaging. (C) Western blots showing higher expression of Myc and Gfi1b in CSCG cells compared with the parental CS cells. The expression level of MycN, Gfi1, and p53 proteins were the same in both cell lines.

    Article Snippet: Cells formed neurospheres on an ultra-low attachment surface (Corning) in Neurobasal-A (NBA) medium containing B-27 and N2 growth supplements (Life Technologies), epidermal growth factor (EGF) (Sigma-Aldrich) and basic fibroblast growth factor (bFGF) 2 (Miltenyi Biotec) (NBA media).

    Techniques: Over Expression, Expressing, Mouse Assay, Imaging, Western Blot

    MusTRD1/BEN represses TFII-I transcriptional activity and promotes TFII-I nuclear exclusion. ( A ) c -fos -luciferase activity of TFII-I and/or MusTRD1/BEN in the presence (filled columns) or in the absence (empty columns) of 25 ng/ml of recombinant human epidermal growth factor. Lanes 1 and 2, empty vector; 3 and 4, TFII-I; 5 and 6, MusTRD1/BEN; 7 and 8, TFII-I + MusTRD1/BEN. ( B ) Cell extracts from A were Western blotted with an anti-MusTRD1/BEN (α-MusTRD1/BEN) antibody and then stripped and reprobed with anti-TFII-I antibody (α-TFII-I). ( C ) Confocal microscopic analysis of cotransfected GFP-MusTRD1/BEN and GST-TFII-I COS7 cells. Cells were stained with anti-TFII-I antibody and a secondary antibody coupled to Alexa 594. Duplicate experiments (either a–c or d–f ) are shown. a and d show GFP-MusTRD1/BEN (green); b and e show TFII-I (red); and c and f are the superimposition of a and b ( c ) or d and e ( f ), and colocalization is in yellow ( a–c , scale bar = 10 μm; d–f , scale bar = 25 μm). Nuclear localization of an ectopic TFII-I (but not ectopic MusTRD1/BEN)-expressing cell is shown by an arrow.

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

    Article Title: Repression of TFII-I-dependent transcription by nuclear exclusion

    doi: 10.1073/pnas.141222298

    Figure Lengend Snippet: MusTRD1/BEN represses TFII-I transcriptional activity and promotes TFII-I nuclear exclusion. ( A ) c -fos -luciferase activity of TFII-I and/or MusTRD1/BEN in the presence (filled columns) or in the absence (empty columns) of 25 ng/ml of recombinant human epidermal growth factor. Lanes 1 and 2, empty vector; 3 and 4, TFII-I; 5 and 6, MusTRD1/BEN; 7 and 8, TFII-I + MusTRD1/BEN. ( B ) Cell extracts from A were Western blotted with an anti-MusTRD1/BEN (α-MusTRD1/BEN) antibody and then stripped and reprobed with anti-TFII-I antibody (α-TFII-I). ( C ) Confocal microscopic analysis of cotransfected GFP-MusTRD1/BEN and GST-TFII-I COS7 cells. Cells were stained with anti-TFII-I antibody and a secondary antibody coupled to Alexa 594. Duplicate experiments (either a–c or d–f ) are shown. a and d show GFP-MusTRD1/BEN (green); b and e show TFII-I (red); and c and f are the superimposition of a and b ( c ) or d and e ( f ), and colocalization is in yellow ( a–c , scale bar = 10 μm; d–f , scale bar = 25 μm). Nuclear localization of an ectopic TFII-I (but not ectopic MusTRD1/BEN)-expressing cell is shown by an arrow.

    Article Snippet: Before harvesting, cells were serum-starved for 12 h and stimulated with 25 ng/ml of recombinant human epidermal growth factor (Sigma) for 4 h. To test for GAL4 transcriptional activity, a GAL4 reporter, pFR-Luc (Stratagene) (200 ng), and pRL-TK (35 ng) were transfected alone or with the GAL4 expression vector pMA242 (200 ng) ( ) and/or pEBG-MusTRD1/BEN (500 ng).

    Techniques: Activity Assay, Luciferase, Recombinant, Plasmid Preparation, Western Blot, Staining, Expressing

    Proliferating HaCaT cells were more sensitive to EISO-induced plasma membrane damage than quiescent cells HaCaT cells were starved in serum-free DMEM for 24 hr. Fresh media supplemented with bovine pituitary extract and epidermal growth factor (A, C, E, G) or not supplemented (B, D, F, H) was added to the cells for 3 hr prior to treatment with EISO. At the indicated times, cells were detached by trypsinization and pooled with floating cells. Cells were pelleted, resuspended in PBS and incubated with fluorescein diacetate and propidium iodide for 15 minutes before analysis by flow cytometry. Using control cells loaded with individual dyes, regions of cells were identified as live cells with uncompromised membranes (R2), live cells with compromised membranes (R3), or severely compromised cells. Serum-starved EISO-treated cells (6 hr; panel D) displayed 2 distinct PI-stained populations of actively cycling cells in G 1  and G 2  phases (R7) that was used to confirm the population of cells with compromised membranes (evidenced by reduced fluorescein retention) but that were still alive.

    Journal: Archives of biochemistry and biophysics

    Article Title: A novel chemopreventive mechanism for a traditional medicine: East Indian sandalwood oil induces autophagy and cell death in proliferating keratinocytes

    doi: 10.1016/j.abb.2014.06.021

    Figure Lengend Snippet: Proliferating HaCaT cells were more sensitive to EISO-induced plasma membrane damage than quiescent cells HaCaT cells were starved in serum-free DMEM for 24 hr. Fresh media supplemented with bovine pituitary extract and epidermal growth factor (A, C, E, G) or not supplemented (B, D, F, H) was added to the cells for 3 hr prior to treatment with EISO. At the indicated times, cells were detached by trypsinization and pooled with floating cells. Cells were pelleted, resuspended in PBS and incubated with fluorescein diacetate and propidium iodide for 15 minutes before analysis by flow cytometry. Using control cells loaded with individual dyes, regions of cells were identified as live cells with uncompromised membranes (R2), live cells with compromised membranes (R3), or severely compromised cells. Serum-starved EISO-treated cells (6 hr; panel D) displayed 2 distinct PI-stained populations of actively cycling cells in G 1 and G 2 phases (R7) that was used to confirm the population of cells with compromised membranes (evidenced by reduced fluorescein retention) but that were still alive.

    Article Snippet: Annexin V antibodies, bovine pituitary extract (BPE) and epidermal growth factor (EGF) were obtained from Life Technologies/Invitrogen (Grand Island, NY).

    Techniques: Incubation, Flow Cytometry, Cytometry, Staining

    UV-induced LC3 processing and expression increased and PARP cleavage decreased in EISO-treated proliferating and quiescent HaCaT cells Serum-starved and BPE/EGF-treated HaCaT cells were treated with EISO for 16 hr. Adherent and floating cells were pooled and lysed in RIPA buffer for analysis of cellular proteins by Western blotting. PARP cleavage was induced by UV-irradiation, as expected, but EISO treatment blocked UV-induced apoptosis in both serum-starved and BPE/EGF treated cells. LC3 expression and processing were increased by UV in both BPE/EGF treated cells and serum-starved cells. EISO treatment increased LC3 processing in both treatment groups, and augmented the UVB response. Proliferating cells produced the most LC3 II after UVB treatment compared to controls.

    Journal: Archives of biochemistry and biophysics

    Article Title: A novel chemopreventive mechanism for a traditional medicine: East Indian sandalwood oil induces autophagy and cell death in proliferating keratinocytes

    doi: 10.1016/j.abb.2014.06.021

    Figure Lengend Snippet: UV-induced LC3 processing and expression increased and PARP cleavage decreased in EISO-treated proliferating and quiescent HaCaT cells Serum-starved and BPE/EGF-treated HaCaT cells were treated with EISO for 16 hr. Adherent and floating cells were pooled and lysed in RIPA buffer for analysis of cellular proteins by Western blotting. PARP cleavage was induced by UV-irradiation, as expected, but EISO treatment blocked UV-induced apoptosis in both serum-starved and BPE/EGF treated cells. LC3 expression and processing were increased by UV in both BPE/EGF treated cells and serum-starved cells. EISO treatment increased LC3 processing in both treatment groups, and augmented the UVB response. Proliferating cells produced the most LC3 II after UVB treatment compared to controls.

    Article Snippet: Annexin V antibodies, bovine pituitary extract (BPE) and epidermal growth factor (EGF) were obtained from Life Technologies/Invitrogen (Grand Island, NY).

    Techniques: Expressing, Western Blot, Irradiation, Produced

    Dual role of p38 in ligand-independent stress-triggered EGFR trafficking in HeLa cells. ( a ) EGFR immunolocalization in untreated versus UVC-exposed cells 1 h post UVC exposure (left), and quantification of surface downregulation in cells exposed to UVC or treated with EGF for the indicated times (right). Arrows indicate perinuclear accumulation of EGFR (green) after UVC exposure. Data are mean±s.e.m. of three independent experiments. ( b ) Untreated or UVC-exposed HeLa cells were fixed after 1 h, or further incubated with the p38 inhibitor SB202190 (SB) for 1 h (left). Untreated or EGF-treated cells were fixed after 30 min, or further incubated with SB for 1 h in the continuous presence of EGF (right). p38 inhibition causes EGFR (green) redistribution to the plasma membrane following UVC, but not EGF exposure. ( c ) Pre-treatment for 30 min with PITSTOPII prevents UVC-induced EGFR (green) internalization (left) but PITSTOPII addition 1 h after UVC-induced EGFR internalization does not affect perinuclear EGFR accumulation or the recycling induced by simultaneous p38 inhibition (right). ( d ) Cells transfected with control or Rab11 siRNA were immunoblotted after 72 h for Rab11 and tubulin to assess knockdown efficiency (top). Rab11 knockdown did not prevent UVC-induced EGFR (green) internalization but prevented EGFR recycling after subsequent SB treatment (bottom). ( e ) Cells transfected with EGFR-GFP were fixed 1 h after UVC exposure and ultrathin cryosections were immuno-labelled for EGFR with 8 nm gold. EGFR-GFP (arrows) is on the limiting membrane and ILVs of MVBs. ( f ) Immunofluorescence analysis of UVC and EGF sequentially exposed HeLa cells (see Methods for experimental details). Red arrows show endosomes containing EGFR (green) and EGF (red). White arrows show EGFR+ve, EGF-ve endosomes, indicating a separate subset of MVBs containing stress-internalized but not EGF-bound EGFR. Scale bar, 5 μm. ( g ) Cells transfected with constitutively active Rab5-Q79L-DsRed were exposed to UVC and incubated for 1 h before treatment with EGF-AlexaFluor 647 (red) for 3 h. Red and white arrows show EGFR+ve/EGF+ve and EGFR+ve/EGF-ve endosomes, respectively. Scale bars, 10 μm for confocal and 100 nm for EM, unless otherwise indicated; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.

    Journal: Nature Communications

    Article Title: WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway

    doi: 10.1038/ncomms8324

    Figure Lengend Snippet: Dual role of p38 in ligand-independent stress-triggered EGFR trafficking in HeLa cells. ( a ) EGFR immunolocalization in untreated versus UVC-exposed cells 1 h post UVC exposure (left), and quantification of surface downregulation in cells exposed to UVC or treated with EGF for the indicated times (right). Arrows indicate perinuclear accumulation of EGFR (green) after UVC exposure. Data are mean±s.e.m. of three independent experiments. ( b ) Untreated or UVC-exposed HeLa cells were fixed after 1 h, or further incubated with the p38 inhibitor SB202190 (SB) for 1 h (left). Untreated or EGF-treated cells were fixed after 30 min, or further incubated with SB for 1 h in the continuous presence of EGF (right). p38 inhibition causes EGFR (green) redistribution to the plasma membrane following UVC, but not EGF exposure. ( c ) Pre-treatment for 30 min with PITSTOPII prevents UVC-induced EGFR (green) internalization (left) but PITSTOPII addition 1 h after UVC-induced EGFR internalization does not affect perinuclear EGFR accumulation or the recycling induced by simultaneous p38 inhibition (right). ( d ) Cells transfected with control or Rab11 siRNA were immunoblotted after 72 h for Rab11 and tubulin to assess knockdown efficiency (top). Rab11 knockdown did not prevent UVC-induced EGFR (green) internalization but prevented EGFR recycling after subsequent SB treatment (bottom). ( e ) Cells transfected with EGFR-GFP were fixed 1 h after UVC exposure and ultrathin cryosections were immuno-labelled for EGFR with 8 nm gold. EGFR-GFP (arrows) is on the limiting membrane and ILVs of MVBs. ( f ) Immunofluorescence analysis of UVC and EGF sequentially exposed HeLa cells (see Methods for experimental details). Red arrows show endosomes containing EGFR (green) and EGF (red). White arrows show EGFR+ve, EGF-ve endosomes, indicating a separate subset of MVBs containing stress-internalized but not EGF-bound EGFR. Scale bar, 5 μm. ( g ) Cells transfected with constitutively active Rab5-Q79L-DsRed were exposed to UVC and incubated for 1 h before treatment with EGF-AlexaFluor 647 (red) for 3 h. Red and white arrows show EGFR+ve/EGF+ve and EGFR+ve/EGF-ve endosomes, respectively. Scale bars, 10 μm for confocal and 100 nm for EM, unless otherwise indicated; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.

    Article Snippet: Reagents and antibodies Reagents were used at the following concentrations: EGF (100 ng ml−1 ), SB202190 (10 μM), dynasore (80 μM), bafilomycin (200 nM) and anisomycin (100 μM) were from Sigma-Aldrich; cisplatin (200 μM) was from Mayne Pharma; PITSTOPII (20 μM) was from Abcam; gefitinib (Iressa/ZD1839, 10 μM) was from Astra Zeneca; EGF-AlexaFluor 488 and 647 (200 ng ml−1 ), Transferrin-AlexaFluor 555 (25 μg ml−1 ) and LysoTracker Red were from Life Technologies; EGF-horseradish peroxidase (EGF-HRP) (100 ng ml−1 ) was made from biotinylated EGF and streptavidin-HRP (from Life Technologies).

    Techniques: Incubation, Inhibition, Transfection, Immunofluorescence, Staining

    Analysis of ligand-activation of intact EGFR mutants. (A) Pools of S2 cells expressing full-length EGFR mutants were analyzed by FACS as described in Materials and Methods. The filled traces represent data from control parental S2 cells treated with a phycoerythrin-conjugated antibody against the EGFR extracellular region, while the open traces represent data from the transfected stable cell pools analyzed in the same fashion. The marked right shift in each case demonstrates that each chimera is expressed appropriately at the cell surface and that our pools sample a wide-range of expression levels. A total of 10,000 cells were analyzed for each FACS analysis. (B) S2 cells stably expressing the noted EGFR mutants were serum starved overnight and then chilled and left unstimulated (−) or treated with 100 ng of EGF/ml on ice for 10 min. Receptor autophosphorylation in normalized whole-cell lysates was analyzed by immunoblotting with antiphosphotyrosine (α-pTyr) antibody (upper blot) and an antibody specific for the EGFR intracellular domain (α-EGFR) (lower blot). Similar studies with TGF-α gave identical results. Studies to assess the dependence on EGF concentration of phosphorylation of the Δ575-584 mutant showed no difference from the wild type.

    Journal: Molecular and Cellular Biology

    Article Title: Epidermal Growth Factor Receptor Dimerization and Activation Require Ligand-Induced Conformational Changes in the Dimer Interface

    doi: 10.1128/MCB.25.17.7734-7742.2005

    Figure Lengend Snippet: Analysis of ligand-activation of intact EGFR mutants. (A) Pools of S2 cells expressing full-length EGFR mutants were analyzed by FACS as described in Materials and Methods. The filled traces represent data from control parental S2 cells treated with a phycoerythrin-conjugated antibody against the EGFR extracellular region, while the open traces represent data from the transfected stable cell pools analyzed in the same fashion. The marked right shift in each case demonstrates that each chimera is expressed appropriately at the cell surface and that our pools sample a wide-range of expression levels. A total of 10,000 cells were analyzed for each FACS analysis. (B) S2 cells stably expressing the noted EGFR mutants were serum starved overnight and then chilled and left unstimulated (−) or treated with 100 ng of EGF/ml on ice for 10 min. Receptor autophosphorylation in normalized whole-cell lysates was analyzed by immunoblotting with antiphosphotyrosine (α-pTyr) antibody (upper blot) and an antibody specific for the EGFR intracellular domain (α-EGFR) (lower blot). Similar studies with TGF-α gave identical results. Studies to assess the dependence on EGF concentration of phosphorylation of the Δ575-584 mutant showed no difference from the wild type.

    Article Snippet: Recombinant EGF and TGF-α were purchased from Chemicon International (formerly Intergen, Inc.) and were used without further purification.

    Techniques: Activation Assay, Expressing, FACS, Transfection, Stable Transfection, Concentration Assay, Mutagenesis

    Effect of BDNF on EGF-induced human NSPC proliferation and migration. Quantitative analysis of human NSPCs proliferation ( A ) and migration ( B ) were determined by BrdU incorporation and Transwell assay following 2 hours treatment of different concentrations of BDNF stimulation with 20 ng/mL EGF or not. (B) Data were expressed as mean ratio compared with the control group; ( C ) Cells incubated with EGF or EGF and BDNF. Nuclei were counter-stained with DAPI; ( D ) Analysis of apoptosis by Flow-cytometry. There was no appreciable change in these groups CON: control (incubated with medium without EGF or BDNF). * p

    Journal: Molecules

    Article Title: BDNF Promotes EGF-Induced Proliferation and Migration of Human Fetal Neural Stem/Progenitor Cells via the PI3K/Akt Pathwa

    doi: 10.3390/molecules161210146

    Figure Lengend Snippet: Effect of BDNF on EGF-induced human NSPC proliferation and migration. Quantitative analysis of human NSPCs proliferation ( A ) and migration ( B ) were determined by BrdU incorporation and Transwell assay following 2 hours treatment of different concentrations of BDNF stimulation with 20 ng/mL EGF or not. (B) Data were expressed as mean ratio compared with the control group; ( C ) Cells incubated with EGF or EGF and BDNF. Nuclei were counter-stained with DAPI; ( D ) Analysis of apoptosis by Flow-cytometry. There was no appreciable change in these groups CON: control (incubated with medium without EGF or BDNF). * p

    Article Snippet: Recombinant EGF and BDNF were obtained from Invitrogen (Carlsbad, CA, USA), and LY294002 was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Migration, BrdU Incorporation Assay, Transwell Assay, Incubation, Staining, Flow Cytometry, Cytometry

    Effects of BDNF on EGF-mediated human NSPC proliferation and migration were abolished by pretreatment with LY294002.EGF: Cells incubated with 20 ng/mL EGF. EGF+BDNF: cells incubated with 20 ng/mL EGF and 50 ng/mL BDNF. LY294002: Cells incubated with LY294002 before exposure to 20 ng/mL EGF and 50 ng/mL BDNF. * p

    Journal: Molecules

    Article Title: BDNF Promotes EGF-Induced Proliferation and Migration of Human Fetal Neural Stem/Progenitor Cells via the PI3K/Akt Pathwa

    doi: 10.3390/molecules161210146

    Figure Lengend Snippet: Effects of BDNF on EGF-mediated human NSPC proliferation and migration were abolished by pretreatment with LY294002.EGF: Cells incubated with 20 ng/mL EGF. EGF+BDNF: cells incubated with 20 ng/mL EGF and 50 ng/mL BDNF. LY294002: Cells incubated with LY294002 before exposure to 20 ng/mL EGF and 50 ng/mL BDNF. * p

    Article Snippet: Recombinant EGF and BDNF were obtained from Invitrogen (Carlsbad, CA, USA), and LY294002 was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Migration, Incubation

    Western blot analysis phosphorylation and expression of AKT. NSPCs were pretreated with LY294002 (20 μM) for 2 hours before exposure to EGF (20 ng/mL) and BDNF (50 ng/mL). ( A ) Western blotting analysis showed increased and decreased activation (phosphorylation) of Akt-1 with BDNF stimulation in NSPCs pretreated with or without LY294002, respectively; ( B ) Data are expreseed as the ratio of phosphorylated to total Akt-1. * p

    Journal: Molecules

    Article Title: BDNF Promotes EGF-Induced Proliferation and Migration of Human Fetal Neural Stem/Progenitor Cells via the PI3K/Akt Pathwa

    doi: 10.3390/molecules161210146

    Figure Lengend Snippet: Western blot analysis phosphorylation and expression of AKT. NSPCs were pretreated with LY294002 (20 μM) for 2 hours before exposure to EGF (20 ng/mL) and BDNF (50 ng/mL). ( A ) Western blotting analysis showed increased and decreased activation (phosphorylation) of Akt-1 with BDNF stimulation in NSPCs pretreated with or without LY294002, respectively; ( B ) Data are expreseed as the ratio of phosphorylated to total Akt-1. * p

    Article Snippet: Recombinant EGF and BDNF were obtained from Invitrogen (Carlsbad, CA, USA), and LY294002 was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Western Blot, Expressing, Activation Assay

    Rif activity was inhibited upon mitogenic stimulation mediated by MAPK or PI3K activation. ( A ) Domain structure of mDia1. Abbreviations: G, GTPase binding region necessary for RhoA binding; DID, diaphanous inhibitory domain; DD, dimerization domain; CC, coiled coil; FH1, formin homology 1 domain; FH2, formin homology 2 domain; DAD, diaphanous autoinhibitory domain; ( B ) pGEX-4T1-RifWT (1–195 aa) and pGEX-4T1-mDia1-G-DID (73–370 aa) recombinant proteins were generated in an E. coli protein expression system and soluble protein was immobilized to glutathione-Sepharose 4B beads for the use in a Rif activation assay. GST-Rif (1–195 aa) was cleaved by thrombin and then the released Rif was loaded with GDP or GTP. mDia1-G-DID beads were then incubated with GDP- or GTP-loaded Rif at 4 °C for 1 h. The beads were washed three times with lysis buffer and eluted with sample buffer for immunoblotting. HEK293 cell lysate was added to mimic the complex intracellular environment; ( C ) HeLa cells were serum starved overnight, then treated with 100 ng/mL Epidermal Growth Factor (EGF), 8 ng/mL Lysophosphatidic Acid (LPA), 1 µg/mL Sphingosine 1-Phosphate (SIP) or 10% serum for 3 min and Rif activation levels then examined by the Rif activation assay described in ( B ); ( D ) HeLa cells were serum starved overnight, then pretreated with or without p38 MAPK, PI3K, or p42/44 MAPK inhibitors for 30 min and then stimulated with 100 ng/mL EGF for 5 min. Rif activation levels were examined by the Rif activation assay. Rif activation levels were quantified by immunoblotting. Data are means ± S.E.M ( n = 3). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Neurotrophin Promotes Neurite Outgrowth by Inhibiting Rif GTPase Activation Downstream of MAPKs and PI3K Signaling

    doi: 10.3390/ijms18010148

    Figure Lengend Snippet: Rif activity was inhibited upon mitogenic stimulation mediated by MAPK or PI3K activation. ( A ) Domain structure of mDia1. Abbreviations: G, GTPase binding region necessary for RhoA binding; DID, diaphanous inhibitory domain; DD, dimerization domain; CC, coiled coil; FH1, formin homology 1 domain; FH2, formin homology 2 domain; DAD, diaphanous autoinhibitory domain; ( B ) pGEX-4T1-RifWT (1–195 aa) and pGEX-4T1-mDia1-G-DID (73–370 aa) recombinant proteins were generated in an E. coli protein expression system and soluble protein was immobilized to glutathione-Sepharose 4B beads for the use in a Rif activation assay. GST-Rif (1–195 aa) was cleaved by thrombin and then the released Rif was loaded with GDP or GTP. mDia1-G-DID beads were then incubated with GDP- or GTP-loaded Rif at 4 °C for 1 h. The beads were washed three times with lysis buffer and eluted with sample buffer for immunoblotting. HEK293 cell lysate was added to mimic the complex intracellular environment; ( C ) HeLa cells were serum starved overnight, then treated with 100 ng/mL Epidermal Growth Factor (EGF), 8 ng/mL Lysophosphatidic Acid (LPA), 1 µg/mL Sphingosine 1-Phosphate (SIP) or 10% serum for 3 min and Rif activation levels then examined by the Rif activation assay described in ( B ); ( D ) HeLa cells were serum starved overnight, then pretreated with or without p38 MAPK, PI3K, or p42/44 MAPK inhibitors for 30 min and then stimulated with 100 ng/mL EGF for 5 min. Rif activation levels were examined by the Rif activation assay. Rif activation levels were quantified by immunoblotting. Data are means ± S.E.M ( n = 3). * p

    Article Snippet: Recombinant human epidermal growth factor (Hu EGF) was from ThermoFisher Scientific.

    Techniques: Activity Assay, Activation Assay, Binding Assay, Recombinant, Generated, Expressing, Incubation, Lysis

    Colocalization of HAOA-coated gold nanoparticles conjugated with EGF. EGF was dyed with Alexa Fluor 647 (red color) and HAOA-coated gold nanoparticles were dyed with Coumarin-6 (green color). The parts were the HAOA-coated gold nanoparticles are associated with EGF, in the same localization, are visible in yellow (scale bar at 5 μm).

    Journal: PLoS ONE

    Article Title: EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy

    doi: 10.1371/journal.pone.0165419

    Figure Lengend Snippet: Colocalization of HAOA-coated gold nanoparticles conjugated with EGF. EGF was dyed with Alexa Fluor 647 (red color) and HAOA-coated gold nanoparticles were dyed with Coumarin-6 (green color). The parts were the HAOA-coated gold nanoparticles are associated with EGF, in the same localization, are visible in yellow (scale bar at 5 μm).

    Article Snippet: Recombinant Human Epidermal Growth Factor (EGF) (PubChem ID: 62253638), Alexa Fluor® 647 and SYPRO® Orange Protein Gel Stain (5,000X Concentrate in DMSO) was purchased from Life Technologies as molecular probes for confocal microscopy and protein conformational studies.

    Techniques:

    EGFR binding assay in A549 cell model, for 1.5 hours in contact with treatment (100X). CN1 corresponds to the non-treated cells, while CN2 shows the exposure to HAOA-coated gold nanoparticles (without any dye). As for the treatment groups: A1) free EGF with Alexa Fluor 647, B1) EGF-conjugated HAOA-coated gold nanoparticles (only EGF is marked with Alexa Fluor 647), and C1) EGF-conjugated HAOA-coated gold nanoparticles (both EGF and HAOA-coated gold nanoparticles are marked with Alexa Fluor 647 and Coumarin-6, respectively). For A2, B2 and C2, anti-EGFR antibodies were added 1 hour before the addition of the tested samples.

    Journal: PLoS ONE

    Article Title: EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy

    doi: 10.1371/journal.pone.0165419

    Figure Lengend Snippet: EGFR binding assay in A549 cell model, for 1.5 hours in contact with treatment (100X). CN1 corresponds to the non-treated cells, while CN2 shows the exposure to HAOA-coated gold nanoparticles (without any dye). As for the treatment groups: A1) free EGF with Alexa Fluor 647, B1) EGF-conjugated HAOA-coated gold nanoparticles (only EGF is marked with Alexa Fluor 647), and C1) EGF-conjugated HAOA-coated gold nanoparticles (both EGF and HAOA-coated gold nanoparticles are marked with Alexa Fluor 647 and Coumarin-6, respectively). For A2, B2 and C2, anti-EGFR antibodies were added 1 hour before the addition of the tested samples.

    Article Snippet: Recombinant Human Epidermal Growth Factor (EGF) (PubChem ID: 62253638), Alexa Fluor® 647 and SYPRO® Orange Protein Gel Stain (5,000X Concentrate in DMSO) was purchased from Life Technologies as molecular probes for confocal microscopy and protein conformational studies.

    Techniques: Binding Assay

    Transactivation of HER2 by leptin . MCF-7 cells were stimulated for 15 min with 100, 200, 500 ng/mL doses of leptin (Lep) or EGF. The expression of Tyr 1248 HER2 (p-HER2), total HER2 levels (HER2) was studied in 100 μg of total proteins by WB with specific Abs, as described in Materials and Methods. The levels of GAPDH in the same blots were assessed to control protein loading. The graph represents levels of Tyr 1248 HER2 relative to total HER2 under different stimuli. Bars represent SE.

    Journal: BMC Cancer

    Article Title: Leptin/HER2 crosstalk in breast cancer: in vitro study and preliminary in vivo analysis

    doi: 10.1186/1471-2407-8-305

    Figure Lengend Snippet: Transactivation of HER2 by leptin . MCF-7 cells were stimulated for 15 min with 100, 200, 500 ng/mL doses of leptin (Lep) or EGF. The expression of Tyr 1248 HER2 (p-HER2), total HER2 levels (HER2) was studied in 100 μg of total proteins by WB with specific Abs, as described in Materials and Methods. The levels of GAPDH in the same blots were assessed to control protein loading. The graph represents levels of Tyr 1248 HER2 relative to total HER2 under different stimuli. Bars represent SE.

    Article Snippet: For growth factor stimulation, the cells (4.5 to 6.0 × 105 cells/100 mm dish) were placed in phenol red-free serum-free medium (SFM) [ ] for 24 h and then stimulated with different doses of leptin (Ob) (R & D systems, Minneapolis, MN, USA) or epidermal growth factor (EGF) (BD Biosciences, Bedford, MA, USA) for 15 min.

    Techniques: Expressing, Western Blot

    Characteristics of OSCs. (a) The representative images of SP cells from K7M2 and MG63 osteosarcoma cell lines. The median value of K7M2 and MG63 SP cells was 1.25% and 1.07%, respectively. n = 5. (b) The representative TEM images of autophagosomes in K7M2 and MG63 SP cells. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (c) Tumor spheres of K7M2 and MG63 osteosarcoma cells after culturing in the serum-free medium DMEM/F12-bFGF-EGF-B27 for 7 days. The parental K7M2 and MG63 cells cultured in DMEM/F12 supplemented with 1% FBS served as a control. Scale bars = 50 μ m. n = 5. (d) Western blot analysis of the pluripotent transcription factors Sox2, Oct4, and Nanog and the autophagy markers LC3, ATG5, and ATG7 in K7M2 and MG63 OSCs. Data are shown as mean ± SD, n = 3. (e) The mRNA expression levels of the pluripotency-associated genes SOX2 , OCT4 , and NANOG and the autophagy-related genes ATG5 and ATG7 . n = 3. (f) Immunofluorescence analysis of autophagy in K7M2 and MG63 SP cells. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 200 μ m. n = 3. (g) Osteogenic and chondrogenic differentiation of K7M2 and MG63 SP cells. Cells differentiated into osteoblasts and chondroblasts were detected by staining with Alizarin Red and Alcian Blue. Scale bars = 100 μ m. n = 3. (h) Flow cytometry-based assay for the pluripotent transcription factors Sox2 and Oct4 and the CSC surface markers CD44, CD105, CD133, and Stro-1 in K7M2 and MG63 SP cells. n = 3. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy

    doi: 10.1155/2019/9290728

    Figure Lengend Snippet: Characteristics of OSCs. (a) The representative images of SP cells from K7M2 and MG63 osteosarcoma cell lines. The median value of K7M2 and MG63 SP cells was 1.25% and 1.07%, respectively. n = 5. (b) The representative TEM images of autophagosomes in K7M2 and MG63 SP cells. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (c) Tumor spheres of K7M2 and MG63 osteosarcoma cells after culturing in the serum-free medium DMEM/F12-bFGF-EGF-B27 for 7 days. The parental K7M2 and MG63 cells cultured in DMEM/F12 supplemented with 1% FBS served as a control. Scale bars = 50 μ m. n = 5. (d) Western blot analysis of the pluripotent transcription factors Sox2, Oct4, and Nanog and the autophagy markers LC3, ATG5, and ATG7 in K7M2 and MG63 OSCs. Data are shown as mean ± SD, n = 3. (e) The mRNA expression levels of the pluripotency-associated genes SOX2 , OCT4 , and NANOG and the autophagy-related genes ATG5 and ATG7 . n = 3. (f) Immunofluorescence analysis of autophagy in K7M2 and MG63 SP cells. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 200 μ m. n = 3. (g) Osteogenic and chondrogenic differentiation of K7M2 and MG63 SP cells. Cells differentiated into osteoblasts and chondroblasts were detected by staining with Alizarin Red and Alcian Blue. Scale bars = 100 μ m. n = 3. (h) Flow cytometry-based assay for the pluripotent transcription factors Sox2 and Oct4 and the CSC surface markers CD44, CD105, CD133, and Stro-1 in K7M2 and MG63 SP cells. n = 3. ∗ P

    Article Snippet: DAPI (BD5010) was obtained from Bioworld Technology, Inc. DMEM (12800082), DMEM/F12 (12400024), Fetal Bovine Serum (FBS, 10099-141), B27 Supplement (17504044), epidermal growth factor (EGF, PHG0311), and basic fibroblast growth factor (bFGF, 13256029) were purchased from Gibco (USA).

    Techniques: Transmission Electron Microscopy, Cell Culture, Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, Cytometry

    KLC have the capacity to stratify. (a) Human skin was decellularized and confirmed complete cell removal by H E staining. (b) ASC were seeded on the epithelial side of human dermal decellularized matrices in the presence or absence of EGF (0.005 µg/mL). Human dermal decellularized matrices seeded with keratinocytes were used as controls. All three groups were kept in the same media conditions and lifted to an air-liquid interphase at the same time. Histological analysis revealed the presence of several layers of KLC with an organization similar to that of keratinocytes (A.1and 3). Surprisingly, ASC without EGF not only did not transdifferentiate into KLC, but instead migrated inside the matrix (A.2). Immunohistochemical analysis revealed a positive staining of cytokeratin-5 (B.3), cytokeratin-10 (C.3) and involucrin (D.3) in stratified KLC matrices comparable to that of the stratified keratinocyte matrix (A, B, C and D.1). No positive staining was found in the ASC seeded matrices (A, B, C and D.2). Images captured using Nikon Digital Sight DS-5M on a Nikon Eclipse 50i microscope at 20x magnification. Scale bars indicate 100 µm (Image representative n = 3 independent experiments).

    Journal: PLoS ONE

    Article Title: Transdifferentiation of Adipose-Derived Stem Cells into Keratinocyte-Like Cells: Engineering a Stratified Epidermis

    doi: 10.1371/journal.pone.0080587

    Figure Lengend Snippet: KLC have the capacity to stratify. (a) Human skin was decellularized and confirmed complete cell removal by H E staining. (b) ASC were seeded on the epithelial side of human dermal decellularized matrices in the presence or absence of EGF (0.005 µg/mL). Human dermal decellularized matrices seeded with keratinocytes were used as controls. All three groups were kept in the same media conditions and lifted to an air-liquid interphase at the same time. Histological analysis revealed the presence of several layers of KLC with an organization similar to that of keratinocytes (A.1and 3). Surprisingly, ASC without EGF not only did not transdifferentiate into KLC, but instead migrated inside the matrix (A.2). Immunohistochemical analysis revealed a positive staining of cytokeratin-5 (B.3), cytokeratin-10 (C.3) and involucrin (D.3) in stratified KLC matrices comparable to that of the stratified keratinocyte matrix (A, B, C and D.1). No positive staining was found in the ASC seeded matrices (A, B, C and D.2). Images captured using Nikon Digital Sight DS-5M on a Nikon Eclipse 50i microscope at 20x magnification. Scale bars indicate 100 µm (Image representative n = 3 independent experiments).

    Article Snippet: The culture media used for all groups was 49.5% KSFM, 49.5% DMEM and 1% antibiotics, however to achieve transdifferentiation to KLC, we either used KCM in a 1∶1 ratio, or human recombinant epidermal growth factor (EGF) (Gibco) (0.005 µg/ml).

    Techniques: Staining, Immunohistochemistry, Microscopy

    Adiponectin inhibits growth factor-mediated proliferation of prostatic epithelial and stromal cells. ( a , b ) CCK-8 proliferation analysis of RWPE1 and WPMY1 cells cultured in regular growth medium, with 0, 1, 5, 10 or 20 μg/ml human recombinant adiponectin treatment. QD 450 values were converted to cell numbers according to the standard curve. (one-way analysis of variance followed by Dunnett’s post-tests; n = 5; *p

    Journal: Scientific Reports

    Article Title: Adiponectin deficiency contributes to the development and progression of benign prostatic hyperplasia in obesity

    doi: 10.1038/srep43771

    Figure Lengend Snippet: Adiponectin inhibits growth factor-mediated proliferation of prostatic epithelial and stromal cells. ( a , b ) CCK-8 proliferation analysis of RWPE1 and WPMY1 cells cultured in regular growth medium, with 0, 1, 5, 10 or 20 μg/ml human recombinant adiponectin treatment. QD 450 values were converted to cell numbers according to the standard curve. (one-way analysis of variance followed by Dunnett’s post-tests; n = 5; *p

    Article Snippet: Recombinant human adiponectin (1065-AP), recombinant mouse adiponectin (5095-AC) and recombinant human epidermal growth factor (236-EG) were obtained from R & D System.

    Techniques: CCK-8 Assay, Cell Culture, Recombinant

    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

    Targeting QDs to EGFR. EGFR labeled with biotinylated EGF (bioEGF), followed by staining with aminoQDs covalently conjugated to streptavidin (not drawn to scale).

    Journal: Journal of the American Chemical Society

    Article Title: Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging

    doi: 10.1021/ja076069p

    Figure Lengend Snippet: Targeting QDs to EGFR. EGFR labeled with biotinylated EGF (bioEGF), followed by staining with aminoQDs covalently conjugated to streptavidin (not drawn to scale).

    Article Snippet: The next day cells were incubated in PBS with 5 mM MgCl2 , 0.5% dialyzed casein, and 60 nM biotinylated EGF (biotin-XX-EGF from Invitrogen: human EGF conjugated at a single site to biotin via a long spacer arm) for 5 min at 4 °C, to stop receptor internalization.

    Techniques: Labeling, Staining

    Eupatolide inhibits the migration and invasion of breast cancer cells. (A) MDA-MB-231 and (B) MCF-7 cells were co-treated with either 100 ng/ml of EGF or 50 ng/ml of TGF-β1, as well as 10 µM eupatolide or a control, and the migration of cells was monitored for 24 h using an IncuCyte live-cell imaging system. After 24 h, relative wound density was acquired by IncuCyte. (C and D) MDA-MB-231 cells were seeded into Transwell chambers and stimulated with (C) 100 ng/ml of EGF or (D) 50 ng/ml TGF-β1 independently, or were co-treated with 10 µM eupatolide. The number of invaded cells in the bottom chamber was counted to measure invasion levels. *P

    Journal: Oncology Letters

    Article Title: Eupatolide inhibits the TGF-β1-induced migration of breast cancer cells via downregulation of SMAD3 phosphorylation and transcriptional repression of ALK5

    doi: 10.3892/ol.2017.6957

    Figure Lengend Snippet: Eupatolide inhibits the migration and invasion of breast cancer cells. (A) MDA-MB-231 and (B) MCF-7 cells were co-treated with either 100 ng/ml of EGF or 50 ng/ml of TGF-β1, as well as 10 µM eupatolide or a control, and the migration of cells was monitored for 24 h using an IncuCyte live-cell imaging system. After 24 h, relative wound density was acquired by IncuCyte. (C and D) MDA-MB-231 cells were seeded into Transwell chambers and stimulated with (C) 100 ng/ml of EGF or (D) 50 ng/ml TGF-β1 independently, or were co-treated with 10 µM eupatolide. The number of invaded cells in the bottom chamber was counted to measure invasion levels. *P

    Article Snippet: Human epidermal growth factor (EGF) and human transforming growth factor-β1 (TGF-β1) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

    Techniques: Migration, Multiple Displacement Amplification, Live Cell Imaging