egf  (R&D Systems)

 
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    Name:
    Human EGF Antibody
    Description:
    The Human EGF Antibody from R D Systems is a goat polyclonal antibody to EGF This antibody reacts with human The Human EGF Antibody has been validated for the following applications Western Blot Neutralization
    Catalog Number:
    ab-236-na
    Price:
    359
    Applications:
    Western Blot, Neutralization
    Host:
    Goat
    Purity:
    Protein A or G purified
    Conjugate:
    Unconjugated
    Immunogen:
    E. coli-derived recombinant human EGF, Asn971-Arg1023, Accession # P01133
    Size:
    1 mg
    Category:
    Primary Antibodies
    Isotype:
    IgG
    Buy from Supplier


    Structured Review

    R&D Systems egf
    Human EGF Antibody
    The Human EGF Antibody from R D Systems is a goat polyclonal antibody to EGF This antibody reacts with human The Human EGF Antibody has been validated for the following applications Western Blot Neutralization
    https://www.bioz.com/result/egf/product/R&D Systems
    Average 95 stars, based on 91 article reviews
    Price from $9.99 to $1999.99
    egf - by Bioz Stars, 2020-11
    95/100 stars

    Images

    1) Product Images from "3D spheroid culture enhances survival and therapeutic capacities of MSCs injected into ischemic kidney"

    Article Title: 3D spheroid culture enhances survival and therapeutic capacities of MSCs injected into ischemic kidney

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12651

    Paracrine secretion of human adipose‐derived mesenchymal stem cells ( MSC s) under different culture conditions. The paracrine secretion of cytokines by 2D cultured MSC s, 3D spheroids of MSC s and 3D spheroids‐derived cells was investigated by Western blotting. The levels of VEGF ( A ), basic fibroblast growth factor ( bFGF ) ( B ), epidermal growth factor ( EGF ) ( C ), hepatocyte growth factor ( HGF ) ( D ), insulin‐like growth factor ( IGF ) ( E ) and tumour necrosis factor‐alpha (TNF‐α) stimulated gene/protein 6 ( TSG ‐6) ( F ) was determined. Band intensities were quantified and normalized to the GAPDH internal control. ** P
    Figure Legend Snippet: Paracrine secretion of human adipose‐derived mesenchymal stem cells ( MSC s) under different culture conditions. The paracrine secretion of cytokines by 2D cultured MSC s, 3D spheroids of MSC s and 3D spheroids‐derived cells was investigated by Western blotting. The levels of VEGF ( A ), basic fibroblast growth factor ( bFGF ) ( B ), epidermal growth factor ( EGF ) ( C ), hepatocyte growth factor ( HGF ) ( D ), insulin‐like growth factor ( IGF ) ( E ) and tumour necrosis factor‐alpha (TNF‐α) stimulated gene/protein 6 ( TSG ‐6) ( F ) was determined. Band intensities were quantified and normalized to the GAPDH internal control. ** P

    Techniques Used: Derivative Assay, Cell Culture, Western Blot

    2) Product Images from "TNFR1 Promotes Tumor Necrosis Factor-mediated Mouse Colon Epithelial Cell Survival through RAF Activation of NF-?B *TNFR1 Promotes Tumor Necrosis Factor-mediated Mouse Colon Epithelial Cell Survival through RAF Activation of NF-?B * S⃞"

    Article Title: TNFR1 Promotes Tumor Necrosis Factor-mediated Mouse Colon Epithelial Cell Survival through RAF Activation of NF-?B *TNFR1 Promotes Tumor Necrosis Factor-mediated Mouse Colon Epithelial Cell Survival through RAF Activation of NF-?B * S⃞

    Journal:

    doi: 10.1074/jbc.M801269200

    TNF promotes MAPK activation in a Ras-independent manner. A, YAMC cells were treated with TNF or EGF, and activated Ras was pulled down using Raf-RBD-conjugated agarose beads. Western blot analysis was used to detect GTP-bound Ras or phospho-MAPK and
    Figure Legend Snippet: TNF promotes MAPK activation in a Ras-independent manner. A, YAMC cells were treated with TNF or EGF, and activated Ras was pulled down using Raf-RBD-conjugated agarose beads. Western blot analysis was used to detect GTP-bound Ras or phospho-MAPK and

    Techniques Used: Activation Assay, Western Blot

    TNF stimulates Raf kinase activity. Endogenous Raf was immunoprecipitated ( IP ) from YAMC cells treated with TNF or EGF and incubated with recombinant MEK and ATP in vitro . Western blot analysis was performed for Raf, phospho-MEK, and phospho-MAPK.
    Figure Legend Snippet: TNF stimulates Raf kinase activity. Endogenous Raf was immunoprecipitated ( IP ) from YAMC cells treated with TNF or EGF and incubated with recombinant MEK and ATP in vitro . Western blot analysis was performed for Raf, phospho-MEK, and phospho-MAPK.

    Techniques Used: Activity Assay, Immunoprecipitation, Incubation, Recombinant, In Vitro, Western Blot

    Anti-apoptotic signaling downstream of TNF receptors is MEK-independent. A, YAMC cells were pretreated with DMSO, PD98059, or U0126 for 30 min followed by TNF for 10 min or EGF for 5 min. Western blot analysis was performed to detect phospho-MAPK,
    Figure Legend Snippet: Anti-apoptotic signaling downstream of TNF receptors is MEK-independent. A, YAMC cells were pretreated with DMSO, PD98059, or U0126 for 30 min followed by TNF for 10 min or EGF for 5 min. Western blot analysis was performed to detect phospho-MAPK,

    Techniques Used: Western Blot

    3) Product Images from "Cross Talk between c-Met and Epidermal Growth Factor Receptor during Retinal Pigment Epithelial Wound Healing"

    Article Title: Cross Talk between c-Met and Epidermal Growth Factor Receptor during Retinal Pigment Epithelial Wound Healing

    Journal:

    doi: 10.1167/iovs.06-0560

    Effects of AG1478 on EGFR phosphorylation and wound healing. ( A ) Serum-starved ARPE-19 cell monolayer was pretreated with Tyrphostin AG1478 (1 μ M) for 1 hour and then stimulated with scratch wound by sharkstooth comb, HGF (50 ng/mL), or HB-EGF
    Figure Legend Snippet: Effects of AG1478 on EGFR phosphorylation and wound healing. ( A ) Serum-starved ARPE-19 cell monolayer was pretreated with Tyrphostin AG1478 (1 μ M) for 1 hour and then stimulated with scratch wound by sharkstooth comb, HGF (50 ng/mL), or HB-EGF

    Techniques Used:

    Wounding- and EGFR ligand–induced c-Met ectodomain shedding. Cultured ARPE-19 cells were wounded by sharkstooth comb (W). Unwounded cells were treated with HGF, HB-EGF, or EGF, with untreated cells as the control (C). Cells were then cultured
    Figure Legend Snippet: Wounding- and EGFR ligand–induced c-Met ectodomain shedding. Cultured ARPE-19 cells were wounded by sharkstooth comb (W). Unwounded cells were treated with HGF, HB-EGF, or EGF, with untreated cells as the control (C). Cells were then cultured

    Techniques Used: Cell Culture

    HGF and HB-EGF accelerated RPE wound healing. Serum-starved ARPE-19 cells at confluence were injured with a 10- μ L pipet tip. Wounded cells were allowed to heal for 17 hours in the presence or absence (Basal) of HGF (50 ng/mL) or HB-EGF (50 ng/mL)
    Figure Legend Snippet: HGF and HB-EGF accelerated RPE wound healing. Serum-starved ARPE-19 cells at confluence were injured with a 10- μ L pipet tip. Wounded cells were allowed to heal for 17 hours in the presence or absence (Basal) of HGF (50 ng/mL) or HB-EGF (50 ng/mL)

    Techniques Used:

    Impaired RPE migratory response to HGF by HB-EGF pretreatment. Growth factor–starved ARPE-19 cells underwent trypsin digestion and adjustment of cell numbers. Cells were pretreated with HB-EGF (50 ng/mL) or HB-EGF along with GM6001 (50 μ
    Figure Legend Snippet: Impaired RPE migratory response to HGF by HB-EGF pretreatment. Growth factor–starved ARPE-19 cells underwent trypsin digestion and adjustment of cell numbers. Cells were pretreated with HB-EGF (50 ng/mL) or HB-EGF along with GM6001 (50 μ

    Techniques Used:

    4) Product Images from "Goblet Cell Associated Antigen Passages are Inhibited During Salmonella typhimurium Infection to Prevent Pathogen Dissemination and Limit Responses to Dietary Antigens"

    Article Title: Goblet Cell Associated Antigen Passages are Inhibited During Salmonella typhimurium Infection to Prevent Pathogen Dissemination and Limit Responses to Dietary Antigens

    Journal: Mucosal immunology

    doi: 10.1038/s41385-018-0007-6

    IL-1β inhibits SI GAPs during Salmonella infection (a–c) Enzyme-linked immunosorbent assays for a) phospho-EGFR, b) EGF, and c) IL-1β on SI epithelium from uninfected C57BL/6 mice or C57BL/6 mice infected with 5×10 7 CFU Δ inv G or wildtype Salmonella 2 days earlier. (d) Density of GAPs in C57BL/6 mice one hour after i.p. injection or luminal injection of vehicle or 100 ng recombinant IL-1β. e) Density of SI GAPs in C57BL/6 mice 2 days after oral PBS or 5×10 7 wildtype Salmonella and daily injection with vehicle or 10 μg/kg of pan-caspase inhibitor (Z-VAD-FMK). (f) Density of GAPs in C57BL/6 or IL1r −/− mice, given 5×10 8 CFU of wildtype Salmonella in the SI lumen 1 hr earlier (left panel) or given 5×10 7 CFU wildtype Salmonella orally 2 days earlier. Data presented as the mean ± SEM, *p
    Figure Legend Snippet: IL-1β inhibits SI GAPs during Salmonella infection (a–c) Enzyme-linked immunosorbent assays for a) phospho-EGFR, b) EGF, and c) IL-1β on SI epithelium from uninfected C57BL/6 mice or C57BL/6 mice infected with 5×10 7 CFU Δ inv G or wildtype Salmonella 2 days earlier. (d) Density of GAPs in C57BL/6 mice one hour after i.p. injection or luminal injection of vehicle or 100 ng recombinant IL-1β. e) Density of SI GAPs in C57BL/6 mice 2 days after oral PBS or 5×10 7 wildtype Salmonella and daily injection with vehicle or 10 μg/kg of pan-caspase inhibitor (Z-VAD-FMK). (f) Density of GAPs in C57BL/6 or IL1r −/− mice, given 5×10 8 CFU of wildtype Salmonella in the SI lumen 1 hr earlier (left panel) or given 5×10 7 CFU wildtype Salmonella orally 2 days earlier. Data presented as the mean ± SEM, *p

    Techniques Used: Infection, Mouse Assay, Injection, Recombinant

    5) Product Images from "A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity"

    Article Title: A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity

    Journal: mAbs

    doi: 10.4161/mabs.3.3.15188

    Dual inhibitory activities of EI-04 on EGFR and IGF-1R. (A) Blockade of europium-labeled EGF binding to EGFR-Fc by EI-04 in comparison to various anti-EGFR mAbs. (B) Blockade of a constant level of IGF-1 (25 nM, left part) or IGF-2 (60 nM, right part)
    Figure Legend Snippet: Dual inhibitory activities of EI-04 on EGFR and IGF-1R. (A) Blockade of europium-labeled EGF binding to EGFR-Fc by EI-04 in comparison to various anti-EGFR mAbs. (B) Blockade of a constant level of IGF-1 (25 nM, left part) or IGF-2 (60 nM, right part)

    Techniques Used: Labeling, Binding Assay

    6) Product Images from "Modeling human early otic sensory cell development with induced pluripotent stem cells"

    Article Title: Modeling human early otic sensory cell development with induced pluripotent stem cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198954

    Schematic summary outlining the otic differentiation from hiPSCs in monolayer culture. (A) As a first step (step1), hiPSCs at day 0 were exposed to FGF3 and FGF10 growth factors until day 13 for early otic/placodal induction and, in a second step (step2) were then differentiated into otic sensory cells by exposure to either RA/EGF or DBZ until day 20. (B-D) Morphological characteristics of hiPSC-derived otic progenitor cells after FGF3/10, RA/EGF and DBZ treatments respectively. Scale bars, 200 μm. Abbreviations: DFNB, DMEM/F12 supplemented with N2 and B27; RA, retinoic acid; DBZ, difluoro-benzeneacetamide; EGF, epithelial growth factor; FGF, fibroblast growth factor.
    Figure Legend Snippet: Schematic summary outlining the otic differentiation from hiPSCs in monolayer culture. (A) As a first step (step1), hiPSCs at day 0 were exposed to FGF3 and FGF10 growth factors until day 13 for early otic/placodal induction and, in a second step (step2) were then differentiated into otic sensory cells by exposure to either RA/EGF or DBZ until day 20. (B-D) Morphological characteristics of hiPSC-derived otic progenitor cells after FGF3/10, RA/EGF and DBZ treatments respectively. Scale bars, 200 μm. Abbreviations: DFNB, DMEM/F12 supplemented with N2 and B27; RA, retinoic acid; DBZ, difluoro-benzeneacetamide; EGF, epithelial growth factor; FGF, fibroblast growth factor.

    Techniques Used: Derivative Assay

    7) Product Images from "The TWEAK Receptor Fn14 is a Src-inducible Protein and a Positive Regulator of Src-driven Cell Invasion"

    Article Title: The TWEAK Receptor Fn14 is a Src-inducible Protein and a Positive Regulator of Src-driven Cell Invasion

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-14-0411

    Effect of dasatinib treatment on Fn14 protein levels in EGFR-mutant HCC2279 and H1975 cells and EGF-stimulated, EGFR wild-type A549 cells
    Figure Legend Snippet: Effect of dasatinib treatment on Fn14 protein levels in EGFR-mutant HCC2279 and H1975 cells and EGF-stimulated, EGFR wild-type A549 cells

    Techniques Used: Mutagenesis

    8) Product Images from "Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness In Vitro - Implication for Drug Development"

    Article Title: Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness In Vitro - Implication for Drug Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092248

    Proliferation response to EGF and HGF is altered in 3D compared to 2D. The plot displays the expected growth surface (i.e. the predicted growth response from the model that was estimated from the data) across varying concentrations of EGF and HGF using the baseline growth estimated for Plate 1 ( Figure S3 ). H1975 cells were plated as a monolayer on a flat bottom plate (2D) or a spheroid in a ULA round bottom plate (3D) and were stimulated with HGF and EGF (250 ng/ml with six serial 1∶2 dilutions) at various combinations at day 1 for 48 hours. These concentrations were dosed in either flat bottom or ULA round bottom plate. Growth (RLU values) was measured in each well by Cell Titer-Glo Assay and was normalized in terms of percent control. The expected growth surface was generated from the linear growth model that included a linear and quadratic terms for EGF and HGF, as well as an interaction term between EGF and HGF.
    Figure Legend Snippet: Proliferation response to EGF and HGF is altered in 3D compared to 2D. The plot displays the expected growth surface (i.e. the predicted growth response from the model that was estimated from the data) across varying concentrations of EGF and HGF using the baseline growth estimated for Plate 1 ( Figure S3 ). H1975 cells were plated as a monolayer on a flat bottom plate (2D) or a spheroid in a ULA round bottom plate (3D) and were stimulated with HGF and EGF (250 ng/ml with six serial 1∶2 dilutions) at various combinations at day 1 for 48 hours. These concentrations were dosed in either flat bottom or ULA round bottom plate. Growth (RLU values) was measured in each well by Cell Titer-Glo Assay and was normalized in terms of percent control. The expected growth surface was generated from the linear growth model that included a linear and quadratic terms for EGF and HGF, as well as an interaction term between EGF and HGF.

    Techniques Used: Glo Assay, Generated

    3D tumor spheroid culture alters basal EGFR and cMET phosphorylation and response to ligand stimulation. Day four 2D monolayer cultures and 3D spheroid from eight lung tumor cell lines were stimulated with or without 100/ml of EGF or HGF for 15 minutes. Phosphorylation for EGFR and cMET was determined by MSD assay. N is equal to 4 replicates per condition. T-test comparing basal phosphorylation in 2D verses 3D cultures. (* indicates p
    Figure Legend Snippet: 3D tumor spheroid culture alters basal EGFR and cMET phosphorylation and response to ligand stimulation. Day four 2D monolayer cultures and 3D spheroid from eight lung tumor cell lines were stimulated with or without 100/ml of EGF or HGF for 15 minutes. Phosphorylation for EGFR and cMET was determined by MSD assay. N is equal to 4 replicates per condition. T-test comparing basal phosphorylation in 2D verses 3D cultures. (* indicates p

    Techniques Used:

    9) Product Images from "Epidermal growth factor treatment of female mice that express APOE4 at an age of advanced pathology mitigates behavioral and cerebrovascular dysfunction"

    Article Title: Epidermal growth factor treatment of female mice that express APOE4 at an age of advanced pathology mitigates behavioral and cerebrovascular dysfunction

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2020.e03919

    Aβ and ApoE levels are not modulated by EGF treatment in female E4FAD+ mice. There are no differences in hippocampal levels of A. Aβ40 (SDS: t(14) = 0.212, p = 0.835. FA: t(14) = 0.123, p = 0.904), B. Aβ42 (SDS: t(14) = 0.397, p = 0.698. FA: t(14) = 0.11, p = 0.914) or C. apoE (SDS: t(14) = 0.315, p = 0.734. FA: t(14) = 0.945, p = 0.361) levels in EGF-treated E4FAD+ mice compared to vehicle treatment when assessed by ELISA. Data expressed as mean +/- SEM. p > 0.05 by Student's t-test. n = 13 (vehicle-treated E4FAD- mice), 12 (EGF-treated E4FAD- mice), 7 (vehicle-treated E4FAD+ mice) and 9 (EGF-treated E4FAD+ mice).
    Figure Legend Snippet: Aβ and ApoE levels are not modulated by EGF treatment in female E4FAD+ mice. There are no differences in hippocampal levels of A. Aβ40 (SDS: t(14) = 0.212, p = 0.835. FA: t(14) = 0.123, p = 0.904), B. Aβ42 (SDS: t(14) = 0.397, p = 0.698. FA: t(14) = 0.11, p = 0.914) or C. apoE (SDS: t(14) = 0.315, p = 0.734. FA: t(14) = 0.945, p = 0.361) levels in EGF-treated E4FAD+ mice compared to vehicle treatment when assessed by ELISA. Data expressed as mean +/- SEM. p > 0.05 by Student's t-test. n = 13 (vehicle-treated E4FAD- mice), 12 (EGF-treated E4FAD- mice), 7 (vehicle-treated E4FAD+ mice) and 9 (EGF-treated E4FAD+ mice).

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    10) Product Images from "Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial-to-mesenchymal transition and immune modulation"

    Article Title: Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial-to-mesenchymal transition and immune modulation

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2012.01610.x

    Effect of bFGF, EGF and ascorbic acid 2-phosphate on HGF secretion by MSCs. ( A ) MSCs were incubated without or with the indicated concentrations of bFGF, ( B ) presence of 10 ng/ml bFGF with indicated concentration of EGF ( C ) and medium supplemented with 10 ng/ml bFGF, 10 ng/ml EGF in the absence or presence of ascorbic acid 2-phosphate for 72 hrs. HGF in the conditioned medium was determined by an ELISA. The values represent the mean ± S.D. MSCs secreted significantly increased HGF in medium supplemented with bFGF, EGF and ascorbic acid 2-phosphate ( P
    Figure Legend Snippet: Effect of bFGF, EGF and ascorbic acid 2-phosphate on HGF secretion by MSCs. ( A ) MSCs were incubated without or with the indicated concentrations of bFGF, ( B ) presence of 10 ng/ml bFGF with indicated concentration of EGF ( C ) and medium supplemented with 10 ng/ml bFGF, 10 ng/ml EGF in the absence or presence of ascorbic acid 2-phosphate for 72 hrs. HGF in the conditioned medium was determined by an ELISA. The values represent the mean ± S.D. MSCs secreted significantly increased HGF in medium supplemented with bFGF, EGF and ascorbic acid 2-phosphate ( P

    Techniques Used: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    11) Product Images from "A combination of Wnt and growth factor signaling induces Arl4c expression to form epithelial tubular structures"

    Article Title: A combination of Wnt and growth factor signaling induces Arl4c expression to form epithelial tubular structures

    Journal: The EMBO Journal

    doi: 10.1002/embj.201386942

    Wnt3a and epidermal growth factor (Wnt3a/EGF) induces nuclear localization of YAP/TAZ during tube formation
    Figure Legend Snippet: Wnt3a and epidermal growth factor (Wnt3a/EGF) induces nuclear localization of YAP/TAZ during tube formation

    Techniques Used:

    Wnt3a and epidermal growth factor (Wnt3a/EGF) induces Arl4c expression through β-catenin and MAP kinase (MAPK) pathways
    Figure Legend Snippet: Wnt3a and epidermal growth factor (Wnt3a/EGF) induces Arl4c expression through β-catenin and MAP kinase (MAPK) pathways

    Techniques Used: Expressing

    12) Product Images from "RNA Interference Screen Identifies Abl Kinase and PDGFR Signaling in Chlamydia trachomatis Entry"

    Article Title: RNA Interference Screen Identifies Abl Kinase and PDGFR Signaling in Chlamydia trachomatis Entry

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000021

    C. trachomatis infection induces phosphorylation of PDGFRβ and recruitment of phospho-PDGFRβ to the site of entry. (A) PDGFRβ was immunoprecipitated from C. trachomatis infected HeLa cells at 1 hpi in the absence or presence of STI571 and immunoblotted with 4G10 antibody. As a positive control, cells were stimulated with 100 ng/ml PDGF-BB. (B) EGFR was immunoprecipitated from C. trachomatis infected HeLa cells at 1 hpi and immunoblotted with 4G10 antibody. As a positive control, cells were stimulated with 100 ng/ml EGF. (C) C. trachomatis infected cells incubated in the absence (panels A–C) or presence of STI571 (panels D–F) or AG1295 (panels G–I) were fixed and stained with anti-phospho-PDGFRβ antibody (green in merge). Bacteria and host DNA were detected using DAPI (blue in merge). The exposure time for each filter of all images was identical.
    Figure Legend Snippet: C. trachomatis infection induces phosphorylation of PDGFRβ and recruitment of phospho-PDGFRβ to the site of entry. (A) PDGFRβ was immunoprecipitated from C. trachomatis infected HeLa cells at 1 hpi in the absence or presence of STI571 and immunoblotted with 4G10 antibody. As a positive control, cells were stimulated with 100 ng/ml PDGF-BB. (B) EGFR was immunoprecipitated from C. trachomatis infected HeLa cells at 1 hpi and immunoblotted with 4G10 antibody. As a positive control, cells were stimulated with 100 ng/ml EGF. (C) C. trachomatis infected cells incubated in the absence (panels A–C) or presence of STI571 (panels D–F) or AG1295 (panels G–I) were fixed and stained with anti-phospho-PDGFRβ antibody (green in merge). Bacteria and host DNA were detected using DAPI (blue in merge). The exposure time for each filter of all images was identical.

    Techniques Used: Infection, Immunoprecipitation, Positive Control, Incubation, Staining

    13) Product Images from "Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts"

    Article Title: Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0040951

    The relationships between cell concentration and HB-EGF, IL-1α, and TGF-β1 concentrations. Fibroblasts (3.0×10 5 cells) and keratinocytes (1.0×10 6 cells) were cultured in 6-well plates. The supernatant and cells that contained both keratinocytes and fibroblasts from 4 wells were harvested at each time point. The cells were then resuspended in 5 ml for counting. The cytokine concentrations were measured by ELISA. (6A) HB-EGF, IL-1α, and TGF-β1 concentrations at different time points. (6B) Total cell concentration (containing both keratinocytes and fibroblasts) at different time points. (6C) The ratio of HB-EGF, IL-1α, and TGF-β1 concentrations over the total cell concentration (×10 6 /ml) at different time points.
    Figure Legend Snippet: The relationships between cell concentration and HB-EGF, IL-1α, and TGF-β1 concentrations. Fibroblasts (3.0×10 5 cells) and keratinocytes (1.0×10 6 cells) were cultured in 6-well plates. The supernatant and cells that contained both keratinocytes and fibroblasts from 4 wells were harvested at each time point. The cells were then resuspended in 5 ml for counting. The cytokine concentrations were measured by ELISA. (6A) HB-EGF, IL-1α, and TGF-β1 concentrations at different time points. (6B) Total cell concentration (containing both keratinocytes and fibroblasts) at different time points. (6C) The ratio of HB-EGF, IL-1α, and TGF-β1 concentrations over the total cell concentration (×10 6 /ml) at different time points.

    Techniques Used: Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    HB EGF, IL-1α and TGFβ1 levels (3a) as well as keratinocyte concentrations (3b) in different cell cultures. 3×10 5 fibroblasts and 106 keratinocytes, respectively stained with PHK2 and PHK26, were cultured or co-cultured for 5 days. The supernatant was collected (Fig. 3a) and HB EGF, IL-1α and TGFβ1 levels were measured with ELISA. Keratinocytes (K) were harvested and resuspended in 2 ml media, and then were counted by Trypan blue exclusion and Flow Cytometry (Fig. 3b). K/F: keratinocytes were cultured with fibroblasts in common 6-well plates; K: keratinocytes alone; F: fibroblasts alone; In the co-cultures, keratinocytes were cultured with fibroblasts in transwells to separate keratinocytes and fibroblasts (K//F), with fibroblasts transfected with IL-1α siRNA (K/Fa) or TGFβ1 siRNA (K/Fb), or with fibroblasts and anti-HB EGF (K/Fc), anti-IL-1α (K/Fd) or anti-TGFβ1 (K/Fe). *P
    Figure Legend Snippet: HB EGF, IL-1α and TGFβ1 levels (3a) as well as keratinocyte concentrations (3b) in different cell cultures. 3×10 5 fibroblasts and 106 keratinocytes, respectively stained with PHK2 and PHK26, were cultured or co-cultured for 5 days. The supernatant was collected (Fig. 3a) and HB EGF, IL-1α and TGFβ1 levels were measured with ELISA. Keratinocytes (K) were harvested and resuspended in 2 ml media, and then were counted by Trypan blue exclusion and Flow Cytometry (Fig. 3b). K/F: keratinocytes were cultured with fibroblasts in common 6-well plates; K: keratinocytes alone; F: fibroblasts alone; In the co-cultures, keratinocytes were cultured with fibroblasts in transwells to separate keratinocytes and fibroblasts (K//F), with fibroblasts transfected with IL-1α siRNA (K/Fa) or TGFβ1 siRNA (K/Fb), or with fibroblasts and anti-HB EGF (K/Fc), anti-IL-1α (K/Fd) or anti-TGFβ1 (K/Fe). *P

    Techniques Used: Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Transfection

    Effects of co-cultures on the growth of Keratinocytes in long term culture. Keratinocytes were cultured with fibroblasts in common 6-well plates (K/F), with Fibroblasts in transwell to separate keratinocytes and fibroblasts (K//F), with 20ng/ml EGF (K+ EGF), with fibroblasts and anti-HB EGF (K/F + anti-HB EGF), anti-IL-1α (K/F + antiIL-1a) or anti-TGFβ1 (K/F antiTGF β1) or keratinocytes alone (K). 6-well-plates were used in the culture and cells were resuspended in 12 ml after harvested. Four cultures in each condition. Keratinocytes and fibroblasts were respectively stained with PKH26 and PKH2 before culture and counted at the end of each culture (stage) by Trypan Blue exclusion and Flow Cytometry.
    Figure Legend Snippet: Effects of co-cultures on the growth of Keratinocytes in long term culture. Keratinocytes were cultured with fibroblasts in common 6-well plates (K/F), with Fibroblasts in transwell to separate keratinocytes and fibroblasts (K//F), with 20ng/ml EGF (K+ EGF), with fibroblasts and anti-HB EGF (K/F + anti-HB EGF), anti-IL-1α (K/F + antiIL-1a) or anti-TGFβ1 (K/F antiTGF β1) or keratinocytes alone (K). 6-well-plates were used in the culture and cells were resuspended in 12 ml after harvested. Four cultures in each condition. Keratinocytes and fibroblasts were respectively stained with PKH26 and PKH2 before culture and counted at the end of each culture (stage) by Trypan Blue exclusion and Flow Cytometry.

    Techniques Used: Cell Culture, Staining, Flow Cytometry, Cytometry

    Effects of different cytokines on the growth of Keratinocytes. Keratinocytes and fibroblasts were respectively stained with PHK26 and PHK2 before co-culture. Keratinocytes were cultured with fibroblasts (K/F), or with 10 ng/ml, 20 ng/ml and 40 ng/ml of HB EGF (K h10, K h20 and Kh40), IL-1α (Ki10, K i20 and K i40) or TGFβ1 (K t10, K t20 and K t40), or keratinocytes alone (K). 6-well-plates were used in the culture and cells were resuspended in 10 ml after harvested on day 5 following the culture. Four cultures in each condition. Keratinocytes were counted by Trypan Blue exclusion and Flow Cytometry.
    Figure Legend Snippet: Effects of different cytokines on the growth of Keratinocytes. Keratinocytes and fibroblasts were respectively stained with PHK26 and PHK2 before co-culture. Keratinocytes were cultured with fibroblasts (K/F), or with 10 ng/ml, 20 ng/ml and 40 ng/ml of HB EGF (K h10, K h20 and Kh40), IL-1α (Ki10, K i20 and K i40) or TGFβ1 (K t10, K t20 and K t40), or keratinocytes alone (K). 6-well-plates were used in the culture and cells were resuspended in 10 ml after harvested on day 5 following the culture. Four cultures in each condition. Keratinocytes were counted by Trypan Blue exclusion and Flow Cytometry.

    Techniques Used: Staining, Co-Culture Assay, Cell Culture, Flow Cytometry, Cytometry

    The effects of co-culture on the migration of keratinocytes and fibroblasts. Keratinocytes (1.0×10 6 cells) were cultured with pre-seeded 3×10 5 fibroblasts in common 6-well plates (K/F), with fibroblasts in a transwell to separate keratinocytes and fibroblasts (K//F), or with fibroblasts and anti-IL-1α (K/Fa), anti-TGFβ1 (K/Fb), or anti-HB EGF (K/Fc). Keratinocytes alone (K) and fibroblasts alone (F) were cultured as controls. All cells were treated with Mitomycin C and the fibroblasts in K/Fp were also treated with 1% paraformaldehyde PBS. 2 days after keratinocytes were seeded, the coverslips were transferred to new wells for additional analysis. The 6-well-plates containing cells on coverslips were used for additional analysis. After culturing the cells for 5 days, the coverslips were removed and the cells in the wells were trypsinized and resuspended in 1 ml for counting. Six cultures were analyzed for each condition. Keratinocytes and fibroblasts were stained with PKH26 and PKH2, respectively, before culturing the cells, and then counted by Trypan Blue cell exclusion and flow cytometry after culture. *P
    Figure Legend Snippet: The effects of co-culture on the migration of keratinocytes and fibroblasts. Keratinocytes (1.0×10 6 cells) were cultured with pre-seeded 3×10 5 fibroblasts in common 6-well plates (K/F), with fibroblasts in a transwell to separate keratinocytes and fibroblasts (K//F), or with fibroblasts and anti-IL-1α (K/Fa), anti-TGFβ1 (K/Fb), or anti-HB EGF (K/Fc). Keratinocytes alone (K) and fibroblasts alone (F) were cultured as controls. All cells were treated with Mitomycin C and the fibroblasts in K/Fp were also treated with 1% paraformaldehyde PBS. 2 days after keratinocytes were seeded, the coverslips were transferred to new wells for additional analysis. The 6-well-plates containing cells on coverslips were used for additional analysis. After culturing the cells for 5 days, the coverslips were removed and the cells in the wells were trypsinized and resuspended in 1 ml for counting. Six cultures were analyzed for each condition. Keratinocytes and fibroblasts were stained with PKH26 and PKH2, respectively, before culturing the cells, and then counted by Trypan Blue cell exclusion and flow cytometry after culture. *P

    Techniques Used: Co-Culture Assay, Migration, Cell Culture, Staining, Flow Cytometry, Cytometry

    The relationships between cell concentration and HB-EGF, IL-1α, and TGF-β1 concentrations. Fibroblasts (3.0×10 5 cells) and keratinocytes (1.0×10 6 cells) were cultured in 6-well plates. The supernatant and cells that contained both keratinocytes and fibroblasts from 4 wells were harvested at each time point. The cells were then resuspended in 5 ml for counting. The cytokine concentrations were measured by ELISA. (6A) HB-EGF, IL-1α, and TGF-β1 concentrations at different time points. (6B) Total cell concentration (containing both keratinocytes and fibroblasts) at different time points. (6C) The ratio of HB-EGF, IL-1α, and TGF-β1 concentrations over the total cell concentration (×10 6 /ml) at different time points.
    Figure Legend Snippet: The relationships between cell concentration and HB-EGF, IL-1α, and TGF-β1 concentrations. Fibroblasts (3.0×10 5 cells) and keratinocytes (1.0×10 6 cells) were cultured in 6-well plates. The supernatant and cells that contained both keratinocytes and fibroblasts from 4 wells were harvested at each time point. The cells were then resuspended in 5 ml for counting. The cytokine concentrations were measured by ELISA. (6A) HB-EGF, IL-1α, and TGF-β1 concentrations at different time points. (6B) Total cell concentration (containing both keratinocytes and fibroblasts) at different time points. (6C) The ratio of HB-EGF, IL-1α, and TGF-β1 concentrations over the total cell concentration (×10 6 /ml) at different time points.

    Techniques Used: Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    HB EGF, IL-1α and TGFβ1 levels (3a) as well as keratinocyte concentrations (3b) in different cell cultures. 3×10 5 fibroblasts and 106 keratinocytes, respectively stained with PHK2 and PHK26, were cultured or co-cultured for 5 days. The supernatant was collected (Fig. 3a) and HB EGF, IL-1α and TGFβ1 levels were measured with ELISA. Keratinocytes (K) were harvested and resuspended in 2 ml media, and then were counted by Trypan blue exclusion and Flow Cytometry (Fig. 3b). K/F: keratinocytes were cultured with fibroblasts in common 6-well plates; K: keratinocytes alone; F: fibroblasts alone; In the co-cultures, keratinocytes were cultured with fibroblasts in transwells to separate keratinocytes and fibroblasts (K//F), with fibroblasts transfected with IL-1α siRNA (K/Fa) or TGFβ1 siRNA (K/Fb), or with fibroblasts and anti-HB EGF (K/Fc), anti-IL-1α (K/Fd) or anti-TGFβ1 (K/Fe). *P
    Figure Legend Snippet: HB EGF, IL-1α and TGFβ1 levels (3a) as well as keratinocyte concentrations (3b) in different cell cultures. 3×10 5 fibroblasts and 106 keratinocytes, respectively stained with PHK2 and PHK26, were cultured or co-cultured for 5 days. The supernatant was collected (Fig. 3a) and HB EGF, IL-1α and TGFβ1 levels were measured with ELISA. Keratinocytes (K) were harvested and resuspended in 2 ml media, and then were counted by Trypan blue exclusion and Flow Cytometry (Fig. 3b). K/F: keratinocytes were cultured with fibroblasts in common 6-well plates; K: keratinocytes alone; F: fibroblasts alone; In the co-cultures, keratinocytes were cultured with fibroblasts in transwells to separate keratinocytes and fibroblasts (K//F), with fibroblasts transfected with IL-1α siRNA (K/Fa) or TGFβ1 siRNA (K/Fb), or with fibroblasts and anti-HB EGF (K/Fc), anti-IL-1α (K/Fd) or anti-TGFβ1 (K/Fe). *P

    Techniques Used: Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Transfection

    14) Product Images from "Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis"

    Article Title: Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200910126

    Expression and activation of FGFR1IIIb and FGFR2IIIb in the skin of control and K5-R1/R2 mice. (A) The expression pattern of FGF7, FGF10, and FGF22 in the skin is shown schematically. FGF7 and FGF10 are expressed by fibroblasts of the dermis and the dermal papilla (DP) of the hair follicles and by epidermal γδ T cells. FGF22 is expressed by keratinocytes. These FGFs activate FGFR1IIIb and FGFR2IIIb on keratinocytes. Bar, 50 µm. (B) RNA from P0, P12, and P18 back skin epidermis of control and K5-R1/R2 mice was analyzed by real-time RT-PCR for the levels of Fgfr1 and Fgfr2 mRNAs. Error bars indicate mean ± SD. n = 3 K5-R1/R2 mice and 2 control mice at P0, n = 5 mice per genotype for P12 and P18. Glyceraldehyde 3-phosphate dehydrogenase ( Gapdh ) mRNA was used for normalization. Data are indicated as the percentage of control. (C) RNA was isolated from the epidermis of adult K5-R1/R2 mice and age-matched control mice. RNA from mouse liver was used as a positive control for FGFR4. Samples of 20 µg of RNA were analyzed by an RNase protection assay for expression of FGFR3, FGFR4, or GAPDH. b, bases. (D) Primary keratinocytes from control and K5-R1/R2 mice were grown to confluency, serum-starved, and treated for 10 min with FGF7, FGF10, EGF, or medium without growth factors (medium). Lysates were analyzed by Western blotting using antibodies against total and phosphorylated signaling proteins or lamin A (loading control).
    Figure Legend Snippet: Expression and activation of FGFR1IIIb and FGFR2IIIb in the skin of control and K5-R1/R2 mice. (A) The expression pattern of FGF7, FGF10, and FGF22 in the skin is shown schematically. FGF7 and FGF10 are expressed by fibroblasts of the dermis and the dermal papilla (DP) of the hair follicles and by epidermal γδ T cells. FGF22 is expressed by keratinocytes. These FGFs activate FGFR1IIIb and FGFR2IIIb on keratinocytes. Bar, 50 µm. (B) RNA from P0, P12, and P18 back skin epidermis of control and K5-R1/R2 mice was analyzed by real-time RT-PCR for the levels of Fgfr1 and Fgfr2 mRNAs. Error bars indicate mean ± SD. n = 3 K5-R1/R2 mice and 2 control mice at P0, n = 5 mice per genotype for P12 and P18. Glyceraldehyde 3-phosphate dehydrogenase ( Gapdh ) mRNA was used for normalization. Data are indicated as the percentage of control. (C) RNA was isolated from the epidermis of adult K5-R1/R2 mice and age-matched control mice. RNA from mouse liver was used as a positive control for FGFR4. Samples of 20 µg of RNA were analyzed by an RNase protection assay for expression of FGFR3, FGFR4, or GAPDH. b, bases. (D) Primary keratinocytes from control and K5-R1/R2 mice were grown to confluency, serum-starved, and treated for 10 min with FGF7, FGF10, EGF, or medium without growth factors (medium). Lysates were analyzed by Western blotting using antibodies against total and phosphorylated signaling proteins or lamin A (loading control).

    Techniques Used: Expressing, Activation Assay, Mouse Assay, Quantitative RT-PCR, Isolation, Positive Control, Rnase Protection Assay, Western Blot

    15) Product Images from "Generation of enterocyte-like cells from human induced pluripotent stem cells for drug absorption and metabolism studies in human small intestine"

    Article Title: Generation of enterocyte-like cells from human induced pluripotent stem cells for drug absorption and metabolism studies in human small intestine

    Journal: Scientific Reports

    doi: 10.1038/srep16479

    Promotion of enterocyte differentiation by combination treatment with three compounds and differentiation period extension. ( A ) The procedure for enterocyte differentiation from human iPS cells by treatment of compounds is presented. From day 19 to 24, the human iPS-derived intestinal cells were treated with the test compounds. ( B ) The gene expression leve ls of the enterocyte marker ANPEP in the test compound-treated human iPS-derived intestinal cells were measured by real-time RT-PCR analysis on day 24. On the y axis, the gene expression levels in “Control (untreated hiPS-ELCs)” were taken as 1.0. ( C ) On day 24, the gene expression levels of the enterocyte marker VILLIN in the PMA, Wortmannin, SB431542, EGF or Wnt3A-treated human iPS-derived intestinal cells were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in “Control” were taken as 1.0. ( D ) Temporal gene expression levels of ANPEP in the human iPS cell-derived intestinal cells (day 24, 29, and 34) were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in Adult Intestine were taken as 1.0. ( E ) The modified enterocyte differentiation protocol is illustrated. ( F ) A morphological image of human iPS-derived enterocyte-like cells is represented. Scale bar represents 100 μm. ( G ) Human iPS cell-derived enterocyte-like cells were assayed for the expression of intestinal marker CDX2 (Red) by immunohistochemistry. Nuclei were stained with DAPI (Blue). Scale bar represents 40 μm. ( H ) Percentages of VILLIN-positive cells in the SB431542, EGF, and Wnt3A-treated enterocyte-like cells were analyzed by flow cytometry analysis on day 24 and 34. Data are represented as the means ± S.E. ( n ≧ 3). Statistical analysis was performed using the unpaired two-tailed student’s t -test. * P
    Figure Legend Snippet: Promotion of enterocyte differentiation by combination treatment with three compounds and differentiation period extension. ( A ) The procedure for enterocyte differentiation from human iPS cells by treatment of compounds is presented. From day 19 to 24, the human iPS-derived intestinal cells were treated with the test compounds. ( B ) The gene expression leve ls of the enterocyte marker ANPEP in the test compound-treated human iPS-derived intestinal cells were measured by real-time RT-PCR analysis on day 24. On the y axis, the gene expression levels in “Control (untreated hiPS-ELCs)” were taken as 1.0. ( C ) On day 24, the gene expression levels of the enterocyte marker VILLIN in the PMA, Wortmannin, SB431542, EGF or Wnt3A-treated human iPS-derived intestinal cells were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in “Control” were taken as 1.0. ( D ) Temporal gene expression levels of ANPEP in the human iPS cell-derived intestinal cells (day 24, 29, and 34) were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in Adult Intestine were taken as 1.0. ( E ) The modified enterocyte differentiation protocol is illustrated. ( F ) A morphological image of human iPS-derived enterocyte-like cells is represented. Scale bar represents 100 μm. ( G ) Human iPS cell-derived enterocyte-like cells were assayed for the expression of intestinal marker CDX2 (Red) by immunohistochemistry. Nuclei were stained with DAPI (Blue). Scale bar represents 40 μm. ( H ) Percentages of VILLIN-positive cells in the SB431542, EGF, and Wnt3A-treated enterocyte-like cells were analyzed by flow cytometry analysis on day 24 and 34. Data are represented as the means ± S.E. ( n ≧ 3). Statistical analysis was performed using the unpaired two-tailed student’s t -test. * P

    Techniques Used: Derivative Assay, Expressing, Marker, Quantitative RT-PCR, Modification, Immunohistochemistry, Staining, Flow Cytometry, Cytometry, Two Tailed Test

    16) Product Images from "TGFβ Signaling Confers Sorafenib Resistance via Induction of Multiple RTKs in Hepatocellular Carcinoma Cells"

    Article Title: TGFβ Signaling Confers Sorafenib Resistance via Induction of Multiple RTKs in Hepatocellular Carcinoma Cells

    Journal: Molecular carcinogenesis

    doi: 10.1002/mc.22592

    TGFβ increases cell sensitivity to multiple growth factors in Huh7 cells. (A) mRNA expression of Huh7 cells treated with 5 ng/mL TGFβ for 48 h. Data are presented as mean±SEM, N=3 biological replicates. (B) Immunoblot of Huh7 cell lysates after cells were treated with 5 ng/mL TGFβ for 48 h, then 20 ng/mL of IGF, EGF, PDGFβ, or FGF1 for 30 min. (C) Proposed model of TGFβ/RTKs/Akt signaling pathways for sorafenib resistance in HCC.
    Figure Legend Snippet: TGFβ increases cell sensitivity to multiple growth factors in Huh7 cells. (A) mRNA expression of Huh7 cells treated with 5 ng/mL TGFβ for 48 h. Data are presented as mean±SEM, N=3 biological replicates. (B) Immunoblot of Huh7 cell lysates after cells were treated with 5 ng/mL TGFβ for 48 h, then 20 ng/mL of IGF, EGF, PDGFβ, or FGF1 for 30 min. (C) Proposed model of TGFβ/RTKs/Akt signaling pathways for sorafenib resistance in HCC.

    Techniques Used: Expressing

    17) Product Images from "Interleukin-2 induces the in vitro maturation of human pluripotent stem cell-derived intestinal organoids"

    Article Title: Interleukin-2 induces the in vitro maturation of human pluripotent stem cell-derived intestinal organoids

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05450-8

    IL-2 activates STAT3 pathway to induce the in vitro maturation of hIOs. a ELISA quantification of IL-2, IL-8, TNFα, IL-22, IL-6, IL-1β, IL-11, EGF, OSM, and IL-10 concentrations in the culture supernatant of stimulated and unstimulated Jurkat T cells. Expression of IL-2R subunits as analyzed by RT-PCR b and Western blot analyses c in co-cultured or IL-2-treated hIOs. d Phosphorylation levels of proteins from multiple signaling pathways in control, co-cultured and IL-2-treated hIOs as reported by a human phospho-kinase array (upper panels). Analysis by functional interaction (FI) network highlighted a significant enrichment of phospho-proteins in the mTOR and STAT3 signaling pathways (lower panels). In the FI network, arrows represent activating/catalyzing connections, solid lines ending in a perpendicular line represent inhibition, solid lines represent complexes or inputs and dashed lines represent predicted FI connections. e Representative images of the morphology of hIOs cultured in the presence of 1 ng/ml IL-2, a key component of the co-culture system, or stimulated Jurkat T conditioned medium (CM) with or without IL-2R-inactivating antibodies (anti-IL-2Rβ, anti-IL-2Rγ c ) for two passages. Quantitative assessment of the size of hIOs (left bottom) and the number of budding structure per hIO (right bottom); n = 12 hIOs per group. f Representative Western blot analysis of p-STAT3, p-AKT and p-P70 S6 Kinase levels in co-cultured and IL-2-treated hIOs. g hIOs cultured in the presence of IL-2 (1 ng/ml) with or without the addition of S3I-201 (5 μM), Stattic (1 μM) or Rapamycin (Rapa; 10 nM). Quantitative assessment of the size of hIOs after one passage (14 days) (left bottom) and the number of budding structure per hIO (right bottom); n = 14 hIOs per group. Data are presented as mean values of replicates ± SEM. *** p
    Figure Legend Snippet: IL-2 activates STAT3 pathway to induce the in vitro maturation of hIOs. a ELISA quantification of IL-2, IL-8, TNFα, IL-22, IL-6, IL-1β, IL-11, EGF, OSM, and IL-10 concentrations in the culture supernatant of stimulated and unstimulated Jurkat T cells. Expression of IL-2R subunits as analyzed by RT-PCR b and Western blot analyses c in co-cultured or IL-2-treated hIOs. d Phosphorylation levels of proteins from multiple signaling pathways in control, co-cultured and IL-2-treated hIOs as reported by a human phospho-kinase array (upper panels). Analysis by functional interaction (FI) network highlighted a significant enrichment of phospho-proteins in the mTOR and STAT3 signaling pathways (lower panels). In the FI network, arrows represent activating/catalyzing connections, solid lines ending in a perpendicular line represent inhibition, solid lines represent complexes or inputs and dashed lines represent predicted FI connections. e Representative images of the morphology of hIOs cultured in the presence of 1 ng/ml IL-2, a key component of the co-culture system, or stimulated Jurkat T conditioned medium (CM) with or without IL-2R-inactivating antibodies (anti-IL-2Rβ, anti-IL-2Rγ c ) for two passages. Quantitative assessment of the size of hIOs (left bottom) and the number of budding structure per hIO (right bottom); n = 12 hIOs per group. f Representative Western blot analysis of p-STAT3, p-AKT and p-P70 S6 Kinase levels in co-cultured and IL-2-treated hIOs. g hIOs cultured in the presence of IL-2 (1 ng/ml) with or without the addition of S3I-201 (5 μM), Stattic (1 μM) or Rapamycin (Rapa; 10 nM). Quantitative assessment of the size of hIOs after one passage (14 days) (left bottom) and the number of budding structure per hIO (right bottom); n = 14 hIOs per group. Data are presented as mean values of replicates ± SEM. *** p

    Techniques Used: In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Functional Assay, Inhibition, Co-Culture Assay

    18) Product Images from "Quantitative Characterization of the Large-Scale Association of ErbB1 and ErbB2 by Flow Cytometric Homo-FRET Measurements"

    Article Title: Quantitative Characterization of the Large-Scale Association of ErbB1 and ErbB2 by Flow Cytometric Homo-FRET Measurements

    Journal:

    doi: 10.1529/biophysj.108.133371

    Monomer percentage and cluster size of ErbB2 in quiescent and stimulated SKBR-3 cells ( A and B ) SKBR-3 cells starved in the presence of 0.1% FCS for 24 h ( solid line ) were stimulated with heregulin ( dashed line ) or EGF ( dotted line ). The distributions
    Figure Legend Snippet: Monomer percentage and cluster size of ErbB2 in quiescent and stimulated SKBR-3 cells ( A and B ) SKBR-3 cells starved in the presence of 0.1% FCS for 24 h ( solid line ) were stimulated with heregulin ( dashed line ) or EGF ( dotted line ). The distributions

    Techniques Used:

    Fitting of the anisotropy model to ErbB1 and ErbB2 anisotropy data. Starved (•) and heregulin-stimulated (▪) SKRB-3 cells were labeled with a mixture of unlabeled and Alexa488-labeled trastuzumab. Starved (◊) and EGF-stimulated
    Figure Legend Snippet: Fitting of the anisotropy model to ErbB1 and ErbB2 anisotropy data. Starved (•) and heregulin-stimulated (▪) SKRB-3 cells were labeled with a mixture of unlabeled and Alexa488-labeled trastuzumab. Starved (◊) and EGF-stimulated

    Techniques Used: Labeling

    19) Product Images from "Predicting ligand-dependent tumors from multi-dimensional signaling features"

    Article Title: Predicting ligand-dependent tumors from multi-dimensional signaling features

    Journal: NPJ Systems Biology and Applications

    doi: 10.1038/s41540-017-0030-3

    Prediction of ligand-induced proliferation using BDTs. a Ratio of true predictions after BDT training with simulated signaling features or receptor expression only, compared to random predictions in the presence of EGF, HRG, IGF or HGF. b For 500 random splits of training and testing cell lines, the BDT outcome is compared to random growth assessment as histogram and cumulative density function, showing the significant improvement due to mechanistic modeling. c Data of in-vitro cell viability screen showing proliferation response (green) or no significant response (red) in different 2D representations of the feature space
    Figure Legend Snippet: Prediction of ligand-induced proliferation using BDTs. a Ratio of true predictions after BDT training with simulated signaling features or receptor expression only, compared to random predictions in the presence of EGF, HRG, IGF or HGF. b For 500 random splits of training and testing cell lines, the BDT outcome is compared to random growth assessment as histogram and cumulative density function, showing the significant improvement due to mechanistic modeling. c Data of in-vitro cell viability screen showing proliferation response (green) or no significant response (red) in different 2D representations of the feature space

    Techniques Used: Expressing, In Vitro

    Importance of receptor surface levels for model response, shown for a selection of calibration cell lines. a Cell line dependent signaling features: Model response to EGF stimulation of two different cell lines resulting in sustained or transient receptor phosphorylation in the BxPc-3 and IGROV-1 cells. Their respective receptor surface levels are shown on the left. The model fits are represented by the colored lines with respective uncertainties (67% confidence intervals) as shades. Data points are shown as dots in the same color. b Model fits for the cell line ACHN stimulated with HGF, EGF and the combination. c Model response to co-stimulation of EGF plus HRG in comparison to the stimulation with EGF, HRG or IGF-1 alone in H322M cells
    Figure Legend Snippet: Importance of receptor surface levels for model response, shown for a selection of calibration cell lines. a Cell line dependent signaling features: Model response to EGF stimulation of two different cell lines resulting in sustained or transient receptor phosphorylation in the BxPc-3 and IGROV-1 cells. Their respective receptor surface levels are shown on the left. The model fits are represented by the colored lines with respective uncertainties (67% confidence intervals) as shades. Data points are shown as dots in the same color. b Model fits for the cell line ACHN stimulated with HGF, EGF and the combination. c Model response to co-stimulation of EGF plus HRG in comparison to the stimulation with EGF, HRG or IGF-1 alone in H322M cells

    Techniques Used: Selection

    20) Product Images from "Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2"

    Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

    Journal: Nature Communications

    doi: 10.1038/ncomms8679

    Phosphorylation of pS-EphA2 is induced by RSK. ( a ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin antibodies. ( b , c ) Whole-cell lysates from HeLa cells pre-treated with LY294002 (10 μM), SB203580 (10 μM), U0126 (5 μM) or BI-D1870 (10 μM) for 30 min and then stimulated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and pS-EphA2 antibodies ( b ), or by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR, pRSK, RSK1, RSK2 and α-tubulin antibodies ( c ). ( d ) HeLa cells were pre-treated with LY294002 or BI-D1870 for 30 min and then stimulated with NaCl (0.3 M), TPA (100 ng ml −1 ) or EGF (10 ng ml −1 ) for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT and α-tubulin. ( e ) T98G and U-87 MG cells starved in FCS-free medium for 24 h were treated with LY294002, MK-2206, U0126 and BI-D1870 for 30 min and then stimulated with 10% FCS for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT, pERK and α-tubulin.
    Figure Legend Snippet: Phosphorylation of pS-EphA2 is induced by RSK. ( a ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin antibodies. ( b , c ) Whole-cell lysates from HeLa cells pre-treated with LY294002 (10 μM), SB203580 (10 μM), U0126 (5 μM) or BI-D1870 (10 μM) for 30 min and then stimulated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and pS-EphA2 antibodies ( b ), or by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR, pRSK, RSK1, RSK2 and α-tubulin antibodies ( c ). ( d ) HeLa cells were pre-treated with LY294002 or BI-D1870 for 30 min and then stimulated with NaCl (0.3 M), TPA (100 ng ml −1 ) or EGF (10 ng ml −1 ) for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT and α-tubulin. ( e ) T98G and U-87 MG cells starved in FCS-free medium for 24 h were treated with LY294002, MK-2206, U0126 and BI-D1870 for 30 min and then stimulated with 10% FCS for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT, pERK and α-tubulin.

    Techniques Used: SDS Page

    21) Product Images from "TLR5 Activation Induces Secretory Interleukin-1 Receptor Antagonist (sIL-1Ra) and Reduces Inflammasome-associated Tissue Damage"

    Article Title: TLR5 Activation Induces Secretory Interleukin-1 Receptor Antagonist (sIL-1Ra) and Reduces Inflammasome-associated Tissue Damage

    Journal: Mucosal immunology

    doi: 10.1038/mi.2010.57

    Secretion of sIL-1Ra by intestinal epithelial cells (IEC) Confluent human model intestinal epithelia (HT29) were stimulated with indicated doses of flagellin (FliC), EGF or 100 IU/mL of human IFNα, IFNβ or IFNγ. Supernatants were taken after 24h of culture or at indicated time periods for IL-8 or sIL-1Ra analysis by ELISA. ( A ) Dose-dependent secretion of sIL-1Ra. ( B ) sIL-1Ra and ( C ) IL-8 time-dependent secretion after stimulation with 100 ng/mL of FliC (Triangle) or control PBS (Circle). ( D ) IL-8 and ( E ) sIL-1Ra secretion after cells were pre-incubated for 30 minutes with DMSO or MG262 in DMSO (10nM) and then stimulated with 100 ng/mL of FliC. ( F ) Induction of sIL-1Ra by type I and type II interferons. ( G ) Induction of sIL-1Ra by EGF.* p
    Figure Legend Snippet: Secretion of sIL-1Ra by intestinal epithelial cells (IEC) Confluent human model intestinal epithelia (HT29) were stimulated with indicated doses of flagellin (FliC), EGF or 100 IU/mL of human IFNα, IFNβ or IFNγ. Supernatants were taken after 24h of culture or at indicated time periods for IL-8 or sIL-1Ra analysis by ELISA. ( A ) Dose-dependent secretion of sIL-1Ra. ( B ) sIL-1Ra and ( C ) IL-8 time-dependent secretion after stimulation with 100 ng/mL of FliC (Triangle) or control PBS (Circle). ( D ) IL-8 and ( E ) sIL-1Ra secretion after cells were pre-incubated for 30 minutes with DMSO or MG262 in DMSO (10nM) and then stimulated with 100 ng/mL of FliC. ( F ) Induction of sIL-1Ra by type I and type II interferons. ( G ) Induction of sIL-1Ra by EGF.* p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation

    22) Product Images from "The Ras-GTPase activity of neurofibromin restrains ERK-dependent FGFR signaling during endochondral bone formation"

    Article Title: The Ras-GTPase activity of neurofibromin restrains ERK-dependent FGFR signaling during endochondral bone formation

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddt162

    Neurofibromin restrains ERK-dependent FGFR signaling in chondrocytes. ( A and B ) Confluent primary chondrocytes from WT or Nf1 Col2 −/− mice were serum-starved and harvested following FGF2 (10 ng/ml, 5 min) (A) or EGF (100 ng/ml, 5 min) (B)
    Figure Legend Snippet: Neurofibromin restrains ERK-dependent FGFR signaling in chondrocytes. ( A and B ) Confluent primary chondrocytes from WT or Nf1 Col2 −/− mice were serum-starved and harvested following FGF2 (10 ng/ml, 5 min) (A) or EGF (100 ng/ml, 5 min) (B)

    Techniques Used: Mouse Assay

    23) Product Images from "A BMP-FGF Morphogen Toggle Switch Drives the Ultrasensitive Expression of Multiple Genes in the Developing Forebrain"

    Article Title: A BMP-FGF Morphogen Toggle Switch Drives the Ultrasensitive Expression of Multiple Genes in the Developing Forebrain

    Journal: PLoS Computational Biology

    doi: 10.1371/journal.pcbi.1003463

    Threshold tuning, ultrasensitivity, and hysteresis experiments implicate CIPF. ( A ) Ttr responses of E12.5 midline cells to BMP4 with and without FGF8 (red and blue lines, respectively; RT-qPCR). FGF8 shifts the (mRNA) Ttr peak from 16 to 32 ng/ml BMP4, but maximal (mRNA) Ttr levels and its apparent ultrasensitivity are unchanged. ( B ) Msx1 responses of E12.5 CPCs to BMP4 with and without FGF2 and EGF (red and blue lines, respectively, curve fit to a Hill equation; RT-qPCR). Msx1 responds linearly to BMP4 (nH = 1.0) in the absence of FGF2 and EGF. With FGF2 and EGF, Msx1 induction becomes ultrasensitive (nH = 3.7) due to its selective suppression at low BMP4 concentrations, as predicted by the CFF and CIPF models. ( C–H ) Washout paradigms and RT-qPCR results for the hysteresis experiments with E12.5 CPCs. For G and H, BMP4 and the BMPR inhibitor LDN193189 were coapplied following BMP4 washout. Msx1 mRNA levels remain relatively high 2 days after BMP4 washout (D). However, after 4 days (F) or after 2 days in the presence of LDN193189 (H), CPCs initially treated with BSA (blue lines) follow different Msx1 induction curves from those treated initially with BMP4 (red lines), thus displaying hysteresis. Error bars represent s.e.m.
    Figure Legend Snippet: Threshold tuning, ultrasensitivity, and hysteresis experiments implicate CIPF. ( A ) Ttr responses of E12.5 midline cells to BMP4 with and without FGF8 (red and blue lines, respectively; RT-qPCR). FGF8 shifts the (mRNA) Ttr peak from 16 to 32 ng/ml BMP4, but maximal (mRNA) Ttr levels and its apparent ultrasensitivity are unchanged. ( B ) Msx1 responses of E12.5 CPCs to BMP4 with and without FGF2 and EGF (red and blue lines, respectively, curve fit to a Hill equation; RT-qPCR). Msx1 responds linearly to BMP4 (nH = 1.0) in the absence of FGF2 and EGF. With FGF2 and EGF, Msx1 induction becomes ultrasensitive (nH = 3.7) due to its selective suppression at low BMP4 concentrations, as predicted by the CFF and CIPF models. ( C–H ) Washout paradigms and RT-qPCR results for the hysteresis experiments with E12.5 CPCs. For G and H, BMP4 and the BMPR inhibitor LDN193189 were coapplied following BMP4 washout. Msx1 mRNA levels remain relatively high 2 days after BMP4 washout (D). However, after 4 days (F) or after 2 days in the presence of LDN193189 (H), CPCs initially treated with BSA (blue lines) follow different Msx1 induction curves from those treated initially with BMP4 (red lines), thus displaying hysteresis. Error bars represent s.e.m.

    Techniques Used: Quantitative RT-PCR

    BMP-FGF CIPF leads to distinct Msx1 and Msx2 EC 50 values. ( A,B ) Msx2 responses in E12.5 CPCs with the same paradigms used for Msx1 in Figure 3 . (A, Left panel) Msx2 induction by BMP4 alone is linear (blue line, nH = 1.2). With FGF2 and EGF, Msx2 induction becomes ultrasensitive (red line, nH = 5.0) similar to Figure 3C . (A, Right panel) In the 2-day washout paradigm with BMPR inhibitor LDN193189, CPCs treated initially with BSA (blue) follow a different Msx2 induction curve compared with those treated initially with BMP4 (red), thus displaying hysteresis. ( B ) Msx1 (blue) and Msx2 (red) responses to BMP4 in the same CPCs, with FGF2 and EGF present. Msx2 has a lower EC 50 (22 ng/ml BMP4, nH = 3.0) than Msx1 (32 ng/ml BMP4, nH = 3.0). ( C,D ) CIPF network changes that affect B T EC 50 values. (C) Networks in which the balance between B I and F I in the CIPF loop is different produce different EC 50 values. (D) Network changes downstream of the CIPF loop, such as B I -to-B T gains, do not shift EC 50 values. See also Figure S6 .
    Figure Legend Snippet: BMP-FGF CIPF leads to distinct Msx1 and Msx2 EC 50 values. ( A,B ) Msx2 responses in E12.5 CPCs with the same paradigms used for Msx1 in Figure 3 . (A, Left panel) Msx2 induction by BMP4 alone is linear (blue line, nH = 1.2). With FGF2 and EGF, Msx2 induction becomes ultrasensitive (red line, nH = 5.0) similar to Figure 3C . (A, Right panel) In the 2-day washout paradigm with BMPR inhibitor LDN193189, CPCs treated initially with BSA (blue) follow a different Msx2 induction curve compared with those treated initially with BMP4 (red), thus displaying hysteresis. ( B ) Msx1 (blue) and Msx2 (red) responses to BMP4 in the same CPCs, with FGF2 and EGF present. Msx2 has a lower EC 50 (22 ng/ml BMP4, nH = 3.0) than Msx1 (32 ng/ml BMP4, nH = 3.0). ( C,D ) CIPF network changes that affect B T EC 50 values. (C) Networks in which the balance between B I and F I in the CIPF loop is different produce different EC 50 values. (D) Network changes downstream of the CIPF loop, such as B I -to-B T gains, do not shift EC 50 values. See also Figure S6 .

    Techniques Used:

    24) Product Images from "The Nf1 Tumor Suppressor Regulates Mouse Skin Wound Healing, Fibroblast Proliferation, and Collagen Deposited by Fibroblasts"

    Article Title: The Nf1 Tumor Suppressor Regulates Mouse Skin Wound Healing, Fibroblast Proliferation, and Collagen Deposited by Fibroblasts

    Journal: The Journal of investigative dermatology

    doi: 10.1046/j.1523-1747.1999.00609.x

    Increased proliferation of Nf1 +/− fibroblasts to serum factors, macrophage-conditioned media, or EGF ( A ) Fibroblast proliferation was analyzed in media with 10% FBS ( solid bars ) or media with 10% FBS + 50% macrophage-conditioned media (MCM; hatched bars ). Each set of bars represents data from fibroblasts from an individual embryo. ( B ) Fibroblast proliferation stimulated by EGF, but not MCM, is inhibited by the EGF receptor antagonist AG1478. Data show results from a single +/+ and +/− embryo and are representative of three individual experiments using cells from different embryos. Data are presented as the mean fold increase in cell number after 8 d in duplicate cultures. Error bars show SEM.
    Figure Legend Snippet: Increased proliferation of Nf1 +/− fibroblasts to serum factors, macrophage-conditioned media, or EGF ( A ) Fibroblast proliferation was analyzed in media with 10% FBS ( solid bars ) or media with 10% FBS + 50% macrophage-conditioned media (MCM; hatched bars ). Each set of bars represents data from fibroblasts from an individual embryo. ( B ) Fibroblast proliferation stimulated by EGF, but not MCM, is inhibited by the EGF receptor antagonist AG1478. Data show results from a single +/+ and +/− embryo and are representative of three individual experiments using cells from different embryos. Data are presented as the mean fold increase in cell number after 8 d in duplicate cultures. Error bars show SEM.

    Techniques Used:

    25) Product Images from "Oct-4 Expression Maintained Cancer Stem-Like Properties in Lung Cancer-Derived CD133-Positive Cells"

    Article Title: Oct-4 Expression Maintained Cancer Stem-Like Properties in Lung Cancer-Derived CD133-Positive Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002637

    Isolation and characterization of lung cancer-derived CD133 + (LC-CD133 + ). (A) Using a magnetic bead method, we sorted CD133 + cells from tissue samples of patients with lung cancer (LC), and characterized them by FACS assay. (B) LC-CD133 + sorted from two patient with No.1 (PLC-CD133 + ) and No.2 (LLC-CD133 + ) were cultured in bFGF and EGF with DMEM serum-free medium. (C) Evaluation of the formation abilities of spheroid-like bodies (SB) from LC-CD133 + and LC-CD133 − under serum-free medium with bFGF EGF. (D) The growth curves of LC-CD133 + and LC-CD133 − were measured by hemocytometer. Bar: 100 µm. Data shown here are the mean±SD of three experiments.
    Figure Legend Snippet: Isolation and characterization of lung cancer-derived CD133 + (LC-CD133 + ). (A) Using a magnetic bead method, we sorted CD133 + cells from tissue samples of patients with lung cancer (LC), and characterized them by FACS assay. (B) LC-CD133 + sorted from two patient with No.1 (PLC-CD133 + ) and No.2 (LLC-CD133 + ) were cultured in bFGF and EGF with DMEM serum-free medium. (C) Evaluation of the formation abilities of spheroid-like bodies (SB) from LC-CD133 + and LC-CD133 − under serum-free medium with bFGF EGF. (D) The growth curves of LC-CD133 + and LC-CD133 − were measured by hemocytometer. Bar: 100 µm. Data shown here are the mean±SD of three experiments.

    Techniques Used: Isolation, Derivative Assay, FACS, Planar Chromatography, Cell Culture

    26) Product Images from "Sgk3 links growth factor signaling to maintenance of progenitor cells in the hair follicle"

    Article Title: Sgk3 links growth factor signaling to maintenance of progenitor cells in the hair follicle

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200504131

    Growth factor signaling is abnormal in Sgk3-null keratinocytes. (A) Treating WT and KO primary keratinocytes with 10 ng/ml EGF reveals normal kinetics of the activation of ERK 1/2 and Akt. (B) In response to 50 ng/ml IGF-1, Sgk3-null keratinocytes exhibit increased activation of ERK 1/2 and Akt. (C) In primary keratinocytes, IGF-1–mediated activation of ERK 1/2 is dependent on PI3K activation, whereas EGF-mediated ERK activation proceeds independently of PI3K. Cells were pretreated with 100 nM wortmannin for 30 min before stimulation. (D) PI3K-dependent IGF-1 activation of ERK does not indicate codependence of PI3K and ERK pathways; phosphorylation of Akt is unaffected by treatment with MEK inhibitor 444937. Cells were pretreated with 1 μM 444937 for 30 min before stimulation. Antibodies are against phosphorylated (activated) or total ERK 1/2 or AKT 1–3 proteins.
    Figure Legend Snippet: Growth factor signaling is abnormal in Sgk3-null keratinocytes. (A) Treating WT and KO primary keratinocytes with 10 ng/ml EGF reveals normal kinetics of the activation of ERK 1/2 and Akt. (B) In response to 50 ng/ml IGF-1, Sgk3-null keratinocytes exhibit increased activation of ERK 1/2 and Akt. (C) In primary keratinocytes, IGF-1–mediated activation of ERK 1/2 is dependent on PI3K activation, whereas EGF-mediated ERK activation proceeds independently of PI3K. Cells were pretreated with 100 nM wortmannin for 30 min before stimulation. (D) PI3K-dependent IGF-1 activation of ERK does not indicate codependence of PI3K and ERK pathways; phosphorylation of Akt is unaffected by treatment with MEK inhibitor 444937. Cells were pretreated with 1 μM 444937 for 30 min before stimulation. Antibodies are against phosphorylated (activated) or total ERK 1/2 or AKT 1–3 proteins.

    Techniques Used: Activation Assay

    Growth factor signaling elements are present in the hair follicle. (A) Schematic depicting EGF and IGF-1 pathways and known effects on the anagen to catagen transition. Note the strong evidence that EGF and EGFR promote catagen, which are consistent with the observation that Pten, which is inhibitory to PI3K, may promote anagen. (B–I) Localization of growth factor signaling in the hair follicle. Phospho-Akt is present strongly in the upper ORS, colocalizing with K5, in both WT and KO skin at P6 (B–E). EGFR is expressed in the ORS, colocalizing with K5, and in the interfollicular epidermis and hair bulb (F–I). Arrowheads indicate colocalization with K5 in the ORS.
    Figure Legend Snippet: Growth factor signaling elements are present in the hair follicle. (A) Schematic depicting EGF and IGF-1 pathways and known effects on the anagen to catagen transition. Note the strong evidence that EGF and EGFR promote catagen, which are consistent with the observation that Pten, which is inhibitory to PI3K, may promote anagen. (B–I) Localization of growth factor signaling in the hair follicle. Phospho-Akt is present strongly in the upper ORS, colocalizing with K5, in both WT and KO skin at P6 (B–E). EGFR is expressed in the ORS, colocalizing with K5, and in the interfollicular epidermis and hair bulb (F–I). Arrowheads indicate colocalization with K5 in the ORS.

    Techniques Used:

    27) Product Images from "Renal Fibrosis "

    Article Title: Renal Fibrosis

    Journal: The American Journal of Pathology

    doi:

    Tubular epithelial cell interactions with collagen types I and IV. MCT cells adhere preferably to type IV collagen (383 ±16.6% compared to uncoated plastic control) than to type I collagen (232.2 ± 20.5%) in cell adhesion assay ( A , left ). After induction of EMT with TGF-β 1 and EGF, MCT cells with a fibroblast-like morphology attached increasingly to collagen type I (396.7 ± 24.3% compared to uncoated plastic control), whereas adhesion to type IV collagen was less abundant (299.5 ± 20.6%) ( A , right ). MCT cells grown in K1 medium adhere strongly to type IV collagen and seem to display a round-shaped, epithelial cell-like, morphology ( B , left ). MCT cells that were pretreated with TGF-β 1 and EGF gain in capacity to attach to type I collagen and appear to have a more spindle-shaped morphology ( B , right ). Cultivation on type I collagen increased FSP-1 expression in MCT cells that were grown in K1 medium (140 ± 9.3% compared to uncoated plastic control), whereas cultivation on type IV collagen had no effect on FSP-1 expression in untreated cells ( C , left ) as was measured by ELISA of cell lysates. When EMT was induced in MCT cells with TGF-β 1 and EGF, cultivation on type I collagen further increased levels of FSP-1 expression (130.6 ± 7.3% compared to uncoated plastic control), whereas coating with type IV collagen decreased levels of FSP-1 expression (58.1 ± 10.4%) and thus stabilized the epithelial phenotype ( C , right ). *, P
    Figure Legend Snippet: Tubular epithelial cell interactions with collagen types I and IV. MCT cells adhere preferably to type IV collagen (383 ±16.6% compared to uncoated plastic control) than to type I collagen (232.2 ± 20.5%) in cell adhesion assay ( A , left ). After induction of EMT with TGF-β 1 and EGF, MCT cells with a fibroblast-like morphology attached increasingly to collagen type I (396.7 ± 24.3% compared to uncoated plastic control), whereas adhesion to type IV collagen was less abundant (299.5 ± 20.6%) ( A , right ). MCT cells grown in K1 medium adhere strongly to type IV collagen and seem to display a round-shaped, epithelial cell-like, morphology ( B , left ). MCT cells that were pretreated with TGF-β 1 and EGF gain in capacity to attach to type I collagen and appear to have a more spindle-shaped morphology ( B , right ). Cultivation on type I collagen increased FSP-1 expression in MCT cells that were grown in K1 medium (140 ± 9.3% compared to uncoated plastic control), whereas cultivation on type IV collagen had no effect on FSP-1 expression in untreated cells ( C , left ) as was measured by ELISA of cell lysates. When EMT was induced in MCT cells with TGF-β 1 and EGF, cultivation on type I collagen further increased levels of FSP-1 expression (130.6 ± 7.3% compared to uncoated plastic control), whereas coating with type IV collagen decreased levels of FSP-1 expression (58.1 ± 10.4%) and thus stabilized the epithelial phenotype ( C , right ). *, P

    Techniques Used: Cell Adhesion Assay, Expressing, Enzyme-linked Immunosorbent Assay

    MCT cells acquire a fibroblast-like morphology after incubation with type IV collagen α1NC1 domain, similar to changes that were observed after incubation with TGF-β 1 and EGF. Tubular epithelial MCT cells display their typical cobblestone-like morphology when cultured in K1 medium ( A ). When medium was supplemented with either soluble type IV collagen α1NC1 ( C ) domain or with 3 ng/ml TGF-β 1 and 10 ng/ml EGF ( D ) cells acquired a typical spindle-shaped morphology, which is typically observed in the induction of EMT. Incubation with type IV collagen 7S domain did not alter the cellular phenotype ( B ). Original magnifications, ×400.
    Figure Legend Snippet: MCT cells acquire a fibroblast-like morphology after incubation with type IV collagen α1NC1 domain, similar to changes that were observed after incubation with TGF-β 1 and EGF. Tubular epithelial MCT cells display their typical cobblestone-like morphology when cultured in K1 medium ( A ). When medium was supplemented with either soluble type IV collagen α1NC1 ( C ) domain or with 3 ng/ml TGF-β 1 and 10 ng/ml EGF ( D ) cells acquired a typical spindle-shaped morphology, which is typically observed in the induction of EMT. Incubation with type IV collagen 7S domain did not alter the cellular phenotype ( B ). Original magnifications, ×400.

    Techniques Used: Incubation, Cell Culture

    TGF-β1 mRNA expression is up-regulated in EMT. Induction of EMT by TGF-β 1 /EGF or with type IV collagen α1NC1 domain results in up-regulation of TGF-β 1 mRNA expression ( A ). Induction with TGF-β 1 /EGF resulted in a peak after 6 hours, whereas after incubation with type IV collagen α1NC1 domains resulted in an up-regulation after 24 hours ( B ) summarizes the densitometric analysis of experiments with TGF-β 1 /EGF ( left ) and with α1NC1 domain ( right ). Treatment of MCT cells with TGF-β 1 and EGF results in an increase in TGF-β 1 mRNA after 6 hours by (291 ± 48% compared to K1 control), and remains elevated after 12 hours (204 ± 34%) and 24 hours (162 ± 18%). Treatment with α1NC1 domain leads to a significant increase in TGF-β 1 mRNA after 24 hours (175.2 ± 24%), however results after 6 hours (110.7 ± 8%) and 12 hours (125.7 ± 9%) were not significant. In solid-phase direct ELISA for FSP-1 ( C ) co-incubation of α1NC1 domain with neutralizing antibodies to TGF-β 1 (MCT NC1 anti-TGF) reduced the increase in FSP-1 expression significantly (158.1 ± 23% compared to K1 control), whereas the addition of neutralizing EGF antibodies (MCT NC1 anti-EGF) had no significant effect (193.5 ± 29%). Addition of neutralizing antibodies to TGF and EGF (MCT TE anti-TE) abolished growth factor-induced EMT (111.8 ± 14%). These results suggest a role for TGF-β 1 autocrine stimulation for mediation of EMT. *, P
    Figure Legend Snippet: TGF-β1 mRNA expression is up-regulated in EMT. Induction of EMT by TGF-β 1 /EGF or with type IV collagen α1NC1 domain results in up-regulation of TGF-β 1 mRNA expression ( A ). Induction with TGF-β 1 /EGF resulted in a peak after 6 hours, whereas after incubation with type IV collagen α1NC1 domains resulted in an up-regulation after 24 hours ( B ) summarizes the densitometric analysis of experiments with TGF-β 1 /EGF ( left ) and with α1NC1 domain ( right ). Treatment of MCT cells with TGF-β 1 and EGF results in an increase in TGF-β 1 mRNA after 6 hours by (291 ± 48% compared to K1 control), and remains elevated after 12 hours (204 ± 34%) and 24 hours (162 ± 18%). Treatment with α1NC1 domain leads to a significant increase in TGF-β 1 mRNA after 24 hours (175.2 ± 24%), however results after 6 hours (110.7 ± 8%) and 12 hours (125.7 ± 9%) were not significant. In solid-phase direct ELISA for FSP-1 ( C ) co-incubation of α1NC1 domain with neutralizing antibodies to TGF-β 1 (MCT NC1 anti-TGF) reduced the increase in FSP-1 expression significantly (158.1 ± 23% compared to K1 control), whereas the addition of neutralizing EGF antibodies (MCT NC1 anti-EGF) had no significant effect (193.5 ± 29%). Addition of neutralizing antibodies to TGF and EGF (MCT TE anti-TE) abolished growth factor-induced EMT (111.8 ± 14%). These results suggest a role for TGF-β 1 autocrine stimulation for mediation of EMT. *, P

    Techniques Used: Expressing, Incubation, Direct ELISA

    Solid phase direct ELISA for FSP-1 and cytokeratin. Treatment of MCT cells with type IV collagen α1NC1 domain resulted in an increase in FSP-1 expression ( A and C ) and a decrease in cytokeratin expression as compared to untreated K1 control ( B and D ). Effects were similar to those that were obtained by stimulation with TGF-β 1 and EGF, which is currently the most established inductor of EMT in vitro . After 48 hours the α1NC1 domain induced an increase in FSP-1 expression (162.7 ± 14.1% compared to K1 control) ( A ) and decreased cytokeratin expression (76.1 ± 5.3% compared to K1 control) ( B ), whereas effects of TGF-β 1 and EGF were 156.7 ± 23.6% and 65.0 ± 4.9%, respectively. Similar results were obtained after 72 hours ( C and D ). Type IV collagen α1NC1 domain induced FSP-1 expression (181.6 ± 23.6% compared to control) ( C ) and a decrease in cytokeratin expression by (61.4 ± 9.6% of K1 control) ( D ). Treatment with TGF-β 1 and EGF for 72 hours resulted in an induction of FSP-1 expression (184.7 ± 28.1%) ( C ) and depression of cytokeratin expression (60.7 ± 10.1%) ( D ). In another series of experiments FSP-1 expression was determined by ELISA to test the specificity of α1NC1 domain-induced effects ( E ). ELISAs for FSP-1 display a strong expression in tubulointerstitial fibroblasts and low expression levels in untreated MCT cells (MCT-K1) that were used as a control. Increased expression of FSP-1 compared to untreated MCT control was detectable after treatment with TGF-β 1 and EGF (259.9 ± 40.2% compared to MCT K1 control) as well as with α1NC1 domain (199.2 ± 33.1%). Incubation with type IV collagen 7S domain had no significant effect on FSP-1 expression levels (105.9 ± 1.4%). Co-incubation of α1NC1 domain with α1NC1 neutralizing antibodies (MCT NC1-abNC1) diminished the increase in FSP-1 expression (112.7 ± 7.2%), whereas 7S neutralizing antibodies (MCT NC1-ab7S) had no effect (184.1 ± 23.7%). *, P
    Figure Legend Snippet: Solid phase direct ELISA for FSP-1 and cytokeratin. Treatment of MCT cells with type IV collagen α1NC1 domain resulted in an increase in FSP-1 expression ( A and C ) and a decrease in cytokeratin expression as compared to untreated K1 control ( B and D ). Effects were similar to those that were obtained by stimulation with TGF-β 1 and EGF, which is currently the most established inductor of EMT in vitro . After 48 hours the α1NC1 domain induced an increase in FSP-1 expression (162.7 ± 14.1% compared to K1 control) ( A ) and decreased cytokeratin expression (76.1 ± 5.3% compared to K1 control) ( B ), whereas effects of TGF-β 1 and EGF were 156.7 ± 23.6% and 65.0 ± 4.9%, respectively. Similar results were obtained after 72 hours ( C and D ). Type IV collagen α1NC1 domain induced FSP-1 expression (181.6 ± 23.6% compared to control) ( C ) and a decrease in cytokeratin expression by (61.4 ± 9.6% of K1 control) ( D ). Treatment with TGF-β 1 and EGF for 72 hours resulted in an induction of FSP-1 expression (184.7 ± 28.1%) ( C ) and depression of cytokeratin expression (60.7 ± 10.1%) ( D ). In another series of experiments FSP-1 expression was determined by ELISA to test the specificity of α1NC1 domain-induced effects ( E ). ELISAs for FSP-1 display a strong expression in tubulointerstitial fibroblasts and low expression levels in untreated MCT cells (MCT-K1) that were used as a control. Increased expression of FSP-1 compared to untreated MCT control was detectable after treatment with TGF-β 1 and EGF (259.9 ± 40.2% compared to MCT K1 control) as well as with α1NC1 domain (199.2 ± 33.1%). Incubation with type IV collagen 7S domain had no significant effect on FSP-1 expression levels (105.9 ± 1.4%). Co-incubation of α1NC1 domain with α1NC1 neutralizing antibodies (MCT NC1-abNC1) diminished the increase in FSP-1 expression (112.7 ± 7.2%), whereas 7S neutralizing antibodies (MCT NC1-ab7S) had no effect (184.1 ± 23.7%). *, P

    Techniques Used: Direct ELISA, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Incubation

    28) Product Images from "Quantitative analysis of the responses of murine bone marrow mesenchymal stem cells to EGF, PDGF-BB and fibronectin by factorial design methodology"

    Article Title: Quantitative analysis of the responses of murine bone marrow mesenchymal stem cells to EGF, PDGF-BB and fibronectin by factorial design methodology

    Journal:

    doi: 10.1007/s10616-008-9172-x

    Analysis of the effects of EGF, PDGF-BB, and FN on BMSCs proliferation from two-level factorial design experiments
    Figure Legend Snippet: Analysis of the effects of EGF, PDGF-BB, and FN on BMSCs proliferation from two-level factorial design experiments

    Techniques Used:

    Morphology of BMSCs cultured in CM with EGF, PDGF-BB, and FN ( a – f ). Cells (Passage 2) were plated at a density of 3 × 10 3 cells/cm 2 and grown for 6 days in CM with the three factors. Pictures were taken
    Figure Legend Snippet: Morphology of BMSCs cultured in CM with EGF, PDGF-BB, and FN ( a – f ). Cells (Passage 2) were plated at a density of 3 × 10 3 cells/cm 2 and grown for 6 days in CM with the three factors. Pictures were taken

    Techniques Used: Cell Culture

    29) Product Images from "The neuroprotective activity of heat-treated human platelet lysate biomaterials manufactured from outdated pathogen-reduced (amotosalen/UVA) platelet concentrates"

    Article Title: The neuroprotective activity of heat-treated human platelet lysate biomaterials manufactured from outdated pathogen-reduced (amotosalen/UVA) platelet concentrates

    Journal: Journal of Biomedical Science

    doi: 10.1186/s12929-019-0579-9

    Total protein content and trophic factors in Intercept-platelet lysates. a Total proteins concentration (mg/ml). Concentrations in ng/ml of ( b ) brain-derived neurotropic factor (BDNF), c epidermal growth factor (EGF); d platelet-derived growth factor (PDGF)-AB, e vascular endothelial growth factor (VEGF). The values are expressed as the mean ± SD. I-PPL and I-HPPL were compared to the standard HPPL. ns: not statically significant. * p
    Figure Legend Snippet: Total protein content and trophic factors in Intercept-platelet lysates. a Total proteins concentration (mg/ml). Concentrations in ng/ml of ( b ) brain-derived neurotropic factor (BDNF), c epidermal growth factor (EGF); d platelet-derived growth factor (PDGF)-AB, e vascular endothelial growth factor (VEGF). The values are expressed as the mean ± SD. I-PPL and I-HPPL were compared to the standard HPPL. ns: not statically significant. * p

    Techniques Used: Concentration Assay, Derivative Assay

    30) Product Images from "TGF-β activates Erk MAP kinase signalling through direct phosphorylation of ShcA"

    Article Title: TGF-β activates Erk MAP kinase signalling through direct phosphorylation of ShcA

    Journal:

    doi: 10.1038/sj.emboj.7601818

    TGF-β induces ShcA phosphorylation on serine and tyrosine. ( A ) Autoradiogram of 3T3-Swiss cells cultured in the presence of 32 P-[PO 4 ] and treated with 4 ng/ml TGF-β, 20 ng/ml EGF, or neither (Ctl) for the indicated times. ( B – D
    Figure Legend Snippet: TGF-β induces ShcA phosphorylation on serine and tyrosine. ( A ) Autoradiogram of 3T3-Swiss cells cultured in the presence of 32 P-[PO 4 ] and treated with 4 ng/ml TGF-β, 20 ng/ml EGF, or neither (Ctl) for the indicated times. ( B – D

    Techniques Used: Cell Culture

    31) Product Images from "Neurogenic Effects of Cell-Free Extracts of Adipose Stem Cells"

    Article Title: Neurogenic Effects of Cell-Free Extracts of Adipose Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0148691

    Amelioration of oxidative stress in neural cells. After attachment of NSCs for 7 days in the absence of EGF and bFGF , 10 mM hydrogen peroxide was added to medium with or without CFE-ASC for 2 days, and optical microscopic pictures were obtained (A). The survival rates of the attached neuronal cell population was obtained using WST-1 cell viability assay kits (B). The numbers of neurites were counted and represented as bar graphs. The data were analyzed using Student’s t-test. All data are represented as the mean ± SD. *p
    Figure Legend Snippet: Amelioration of oxidative stress in neural cells. After attachment of NSCs for 7 days in the absence of EGF and bFGF , 10 mM hydrogen peroxide was added to medium with or without CFE-ASC for 2 days, and optical microscopic pictures were obtained (A). The survival rates of the attached neuronal cell population was obtained using WST-1 cell viability assay kits (B). The numbers of neurites were counted and represented as bar graphs. The data were analyzed using Student’s t-test. All data are represented as the mean ± SD. *p

    Techniques Used: Viability Assay

    Proliferation of NSC by CFE-ASCs. Primary dissociated SVZ-derived NSC were maintained by the neurosphere method. SVZ tissue was isolated and digested from mice. NSCs were maintained in DMEM/F12/B27 with EGF and bFGF, forming neurospheres (A). After neurosphere cell expansion, these spheres were then transferred into growth-factor-free medium with 5% FBS and kept for 10 days. Without growth factors, spheres were dissociated and attached on coated cover glass (B). SVZ-derived NSCs were treated with CFE-ASCs and BrdU for 2 days in the absence of EGF and bFGF. BrdU (red) and DAPI (blue) staining was performed and observed with fluorescence microscope. Microscopic images showed that CFE-ASCs-treated NSCs have more BudU positive cells than vehicle-treated cells (C). BrdU positive cells were counted and normalized with positive DAPI. Relative cell numbers were represented as bar graphs (n = 5 per group) (D). Cell proliferation assays were performed and relative optical densities were represented as bar graphs (n = 4) (E). *p
    Figure Legend Snippet: Proliferation of NSC by CFE-ASCs. Primary dissociated SVZ-derived NSC were maintained by the neurosphere method. SVZ tissue was isolated and digested from mice. NSCs were maintained in DMEM/F12/B27 with EGF and bFGF, forming neurospheres (A). After neurosphere cell expansion, these spheres were then transferred into growth-factor-free medium with 5% FBS and kept for 10 days. Without growth factors, spheres were dissociated and attached on coated cover glass (B). SVZ-derived NSCs were treated with CFE-ASCs and BrdU for 2 days in the absence of EGF and bFGF. BrdU (red) and DAPI (blue) staining was performed and observed with fluorescence microscope. Microscopic images showed that CFE-ASCs-treated NSCs have more BudU positive cells than vehicle-treated cells (C). BrdU positive cells were counted and normalized with positive DAPI. Relative cell numbers were represented as bar graphs (n = 5 per group) (D). Cell proliferation assays were performed and relative optical densities were represented as bar graphs (n = 4) (E). *p

    Techniques Used: Derivative Assay, Isolation, Mouse Assay, Staining, Fluorescence, Microscopy

    Neural-differentiation promoting effects of CFE-ASCs. NSCs were cultured with DMEM/F12 with B27 and 5% FBS without EGF and bFGF. After 6 days, NSCs were treated with CFE-ASCs or vehicle for 2 days and stained with Tuj-1, GFAP and DAPI. Fluorescence microscopic observation showed expression of Tuj-1 (Red) and GFAP (Green) (A). Positive-stained cells were counted and normalized with DAPI (Blue) count. Vehicle and CFE-ASC-treated NSCs showed no significant differences in positive-cell numbers (n = 5) (B). Fluorescence intensities of Tuj-1 and GFAP were calculated and normalized with DAPI. Relative fluorescence intensity was higher in the CFE-ASC-treated group compared with the vehicle group (n = 5) (C). The data were analyzed using Student’s t-test. All data are represented as the mean ± SD. *p
    Figure Legend Snippet: Neural-differentiation promoting effects of CFE-ASCs. NSCs were cultured with DMEM/F12 with B27 and 5% FBS without EGF and bFGF. After 6 days, NSCs were treated with CFE-ASCs or vehicle for 2 days and stained with Tuj-1, GFAP and DAPI. Fluorescence microscopic observation showed expression of Tuj-1 (Red) and GFAP (Green) (A). Positive-stained cells were counted and normalized with DAPI (Blue) count. Vehicle and CFE-ASC-treated NSCs showed no significant differences in positive-cell numbers (n = 5) (B). Fluorescence intensities of Tuj-1 and GFAP were calculated and normalized with DAPI. Relative fluorescence intensity was higher in the CFE-ASC-treated group compared with the vehicle group (n = 5) (C). The data were analyzed using Student’s t-test. All data are represented as the mean ± SD. *p

    Techniques Used: Cell Culture, Staining, Fluorescence, Expressing

    32) Product Images from "Principles of early human development and germ cell program from conserved model systems"

    Article Title: Principles of early human development and germ cell program from conserved model systems

    Journal: Nature

    doi: 10.1038/nature22812

    Chronology of transcription factors expression during hPGC induction a. Schematic of hPGC induction from Pre-ME(12h). b. Images of day 2 and 4 embryoids in response to BMP2 alone or BMP2 with LIF, SCF and EGF(=Cytokines). Notably, BMP2 alone can induce hPGC at almost the same efficiency as the full cytokines, but do not survive during extended culture, as shown previously. c. Immunostaining of embryoids induced with BMP2 alone or BMP2 with LIF, SCF and EGF showing expression of SOX17, BLIMP1 and TFAP2C. Scale bar: 50 µm. d. Proportion of SOX17 +ve cells indicated in Extended Data Fig.7c. e. Immunostaining of embryoids induced with BMP2 alone or BMP2 with LIF, SCF and EGF showing expression of SOX17, BLIMP1 and NANOG. Scale bar: 50 µm. f. Proportion of SOX17+ve cells in Extended Data Fig.7e.
    Figure Legend Snippet: Chronology of transcription factors expression during hPGC induction a. Schematic of hPGC induction from Pre-ME(12h). b. Images of day 2 and 4 embryoids in response to BMP2 alone or BMP2 with LIF, SCF and EGF(=Cytokines). Notably, BMP2 alone can induce hPGC at almost the same efficiency as the full cytokines, but do not survive during extended culture, as shown previously. c. Immunostaining of embryoids induced with BMP2 alone or BMP2 with LIF, SCF and EGF showing expression of SOX17, BLIMP1 and TFAP2C. Scale bar: 50 µm. d. Proportion of SOX17 +ve cells indicated in Extended Data Fig.7c. e. Immunostaining of embryoids induced with BMP2 alone or BMP2 with LIF, SCF and EGF showing expression of SOX17, BLIMP1 and NANOG. Scale bar: 50 µm. f. Proportion of SOX17+ve cells in Extended Data Fig.7e.

    Techniques Used: Expressing, Immunostaining

    33) Product Images from "Wnt/?-catenin signaling is a key downstream mediator of MET signaling in glioblastoma stem cells"

    Article Title: Wnt/?-catenin signaling is a key downstream mediator of MET signaling in glioblastoma stem cells

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/nos299

    Inhibition of GSC clonogenic growth by MET inhibition. The 464T GBM cells were cultured in the EGF/bFGF (A and B) or HGF-containing media (C and D). (A and C) To determine the clonogenicity of GBM cells after MET inhibition, we performed neurosphere-limiting
    Figure Legend Snippet: Inhibition of GSC clonogenic growth by MET inhibition. The 464T GBM cells were cultured in the EGF/bFGF (A and B) or HGF-containing media (C and D). (A and C) To determine the clonogenicity of GBM cells after MET inhibition, we performed neurosphere-limiting

    Techniques Used: Inhibition, Cell Culture

    Enrichment of β-catenin high and TCF4 high/+ cells in the MET high/+ cells. (A) After the culture of the patient GBM cells (131) in EGF/bFGF- and HGF-containing growth medium, the cells were costained with β-catenin and MET antibodies by
    Figure Legend Snippet: Enrichment of β-catenin high and TCF4 high/+ cells in the MET high/+ cells. (A) After the culture of the patient GBM cells (131) in EGF/bFGF- and HGF-containing growth medium, the cells were costained with β-catenin and MET antibodies by

    Techniques Used:

    34) Product Images from "Transforming Growth Factor Alpha (TGF?) Regulates Granulosa Cell Tumor (GCT) Cell Proliferation and Migration through Activation of Multiple Pathways"

    Article Title: Transforming Growth Factor Alpha (TGF?) Regulates Granulosa Cell Tumor (GCT) Cell Proliferation and Migration through Activation of Multiple Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0048299

    Expression of TGFα, EGF and ErbB family receptor mRNA in KGN and COV434 GCT cell lines. SKOV-3 (ErbB2 overexpressing cells) and COV644 cells (ErbB4 negative cells) were used as positive and negative controls. Primary cultures of normal human granulosa cells were used as a positive control for the detection of EGFR and TGFα. β-actin mRNA was used as an internal loading control.
    Figure Legend Snippet: Expression of TGFα, EGF and ErbB family receptor mRNA in KGN and COV434 GCT cell lines. SKOV-3 (ErbB2 overexpressing cells) and COV644 cells (ErbB4 negative cells) were used as positive and negative controls. Primary cultures of normal human granulosa cells were used as a positive control for the detection of EGFR and TGFα. β-actin mRNA was used as an internal loading control.

    Techniques Used: Expressing, Positive Control

    35) Product Images from "Endothelial Cell Capture of Heparin-Binding Growth Factors under Flow"

    Article Title: Endothelial Cell Capture of Heparin-Binding Growth Factors under Flow

    Journal: PLoS Computational Biology

    doi: 10.1371/journal.pcbi.1000971

    EGF and VEGF are retained under flow. (A) EGF (1.49 ng) was injected into the input reservoir, pumped through the system at 0.61 mL/min (1.22 mm/sec), and EGF quantified in the output flow by ELISA. Data shown are from the same cartridge either untreated (○) or enzyme-treated (•). FGF-2 (1.01ng - x) is shown for comparison. (B) VEGF was injected into the input reservoir of untreated (0.95ng - ○) or heparinase-treated (0.98ng -•) cartridges, run through the system at 0.66 mL/min (1.32 mm/sec), and VEGF quantified in the output flow by ELISA. Data are representative of at least three runs quantified in Table 4 .
    Figure Legend Snippet: EGF and VEGF are retained under flow. (A) EGF (1.49 ng) was injected into the input reservoir, pumped through the system at 0.61 mL/min (1.22 mm/sec), and EGF quantified in the output flow by ELISA. Data shown are from the same cartridge either untreated (○) or enzyme-treated (•). FGF-2 (1.01ng - x) is shown for comparison. (B) VEGF was injected into the input reservoir of untreated (0.95ng - ○) or heparinase-treated (0.98ng -•) cartridges, run through the system at 0.66 mL/min (1.32 mm/sec), and VEGF quantified in the output flow by ELISA. Data are representative of at least three runs quantified in Table 4 .

    Techniques Used: Flow Cytometry, Injection, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay

    36) Product Images from "The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix"

    Article Title: The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix

    Journal: The Journal of Cell Biology

    doi:

    HaCaT migration on native type I collagen is MMP dependent. ( A ) HaCaT keratinocytes were grown on type I collagen– coated dishes and treated with or without 30 ng/ml EGF. Collagenase-1 accumulation in the medium was assessed 48 h later by ELISA and normalized to total cellular protein. ( B ) HaCaT cells were plated on collagencoated slides, stimulated with EGF, and 24 h later were processed for in situ hybridization with a collagenase-1 35 S-labeled antisense RNA probe. Only HaCaT cells at the periphery of cell clusters expressed collagenase-1 mRNA. Autoradiographic exposure was 14 d. ( C–E ) HaCaT cells were plated within cloning cylinders on collagen-coated dishes. After 24 h, the cylinders were removed, and the cells were allowed to migrate on collagen alone or in the presence of EGF for 48, 72, or 96 h. Cells were stained, and the area migrated was quantified by scanning densitometry. ( E ) During the initial 24-h culture period, some HaCaTs were treated with 100 mM HU to inhibit EGFmediated proliferation. The cylinders were removed, and the cells were given fresh medium with or without 30 ng/ ml EGF or EGF plus 25 μM peptide hydroxymate inhibitor SC44463. After 96 h, cultures were washed and stained, and migration was quantified by image analysis. Migration data for HaCaT cells pretreated with (+ HU ) or without ( −HU ) HU are shown. The data in D and E are the means ± SD or triplicate wells and are expressed in arbitrary units relative to 0-h controls. ( F ) HaCaT keratinocytes were plated on culture slides coated with a mixture of colloidal gold particles and type I collagen ( Col ) or gelatin ( Gel ). Cells on collagen-coated chambers were treated with 30 ng/ml EGF. To inhibit collagenase-1 activity, cells were treated with (+) or without (−) collagenase-1 affinity-purified antibody or 25 μM SC44463 and were fixed 20 h later. Keratinocyte migration was quantified as described under Materials and Methods, and the data shown are the means ± SEM of duplicate samples from four experiments.
    Figure Legend Snippet: HaCaT migration on native type I collagen is MMP dependent. ( A ) HaCaT keratinocytes were grown on type I collagen– coated dishes and treated with or without 30 ng/ml EGF. Collagenase-1 accumulation in the medium was assessed 48 h later by ELISA and normalized to total cellular protein. ( B ) HaCaT cells were plated on collagencoated slides, stimulated with EGF, and 24 h later were processed for in situ hybridization with a collagenase-1 35 S-labeled antisense RNA probe. Only HaCaT cells at the periphery of cell clusters expressed collagenase-1 mRNA. Autoradiographic exposure was 14 d. ( C–E ) HaCaT cells were plated within cloning cylinders on collagen-coated dishes. After 24 h, the cylinders were removed, and the cells were allowed to migrate on collagen alone or in the presence of EGF for 48, 72, or 96 h. Cells were stained, and the area migrated was quantified by scanning densitometry. ( E ) During the initial 24-h culture period, some HaCaTs were treated with 100 mM HU to inhibit EGFmediated proliferation. The cylinders were removed, and the cells were given fresh medium with or without 30 ng/ ml EGF or EGF plus 25 μM peptide hydroxymate inhibitor SC44463. After 96 h, cultures were washed and stained, and migration was quantified by image analysis. Migration data for HaCaT cells pretreated with (+ HU ) or without ( −HU ) HU are shown. The data in D and E are the means ± SD or triplicate wells and are expressed in arbitrary units relative to 0-h controls. ( F ) HaCaT keratinocytes were plated on culture slides coated with a mixture of colloidal gold particles and type I collagen ( Col ) or gelatin ( Gel ). Cells on collagen-coated chambers were treated with 30 ng/ml EGF. To inhibit collagenase-1 activity, cells were treated with (+) or without (−) collagenase-1 affinity-purified antibody or 25 μM SC44463 and were fixed 20 h later. Keratinocyte migration was quantified as described under Materials and Methods, and the data shown are the means ± SEM of duplicate samples from four experiments.

    Techniques Used: Migration, Enzyme-linked Immunosorbent Assay, In Situ Hybridization, Labeling, Clone Assay, Staining, Activity Assay, Affinity Purification

    37) Product Images from "Growth Factor Signaling in Vitreous Humor-Induced Lens Fiber Differentiation"

    Article Title: Growth Factor Signaling in Vitreous Humor-Induced Lens Fiber Differentiation

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.09-4797

    Explants treated for 5 days with no growth factors ( A , E ) or a combination of different growth factors: 50 ng/mL IGF ( B , F ); 15 ng/mL PDGF ( C , G ); 5 ng/mL EGF ( D , H ); 5 ng/mL FGF ( I , M ), or a combination of a low dose of FGF (5 ng/mL) with IGF (FGF/IGF,
    Figure Legend Snippet: Explants treated for 5 days with no growth factors ( A , E ) or a combination of different growth factors: 50 ng/mL IGF ( B , F ); 15 ng/mL PDGF ( C , G ); 5 ng/mL EGF ( D , H ); 5 ng/mL FGF ( I , M ), or a combination of a low dose of FGF (5 ng/mL) with IGF (FGF/IGF,

    Techniques Used:

    Explants treated with EGF (5 ng/mL) or a low dose of FGF (5 ng/mL)+EGF (5 ng/mL). ( i ) Representative Western blots of explants cultured without growth factor (control) or with EGF ( iA ) from 5 minutes up to 24 hours, assayed for phosphorylated Akt ( top
    Figure Legend Snippet: Explants treated with EGF (5 ng/mL) or a low dose of FGF (5 ng/mL)+EGF (5 ng/mL). ( i ) Representative Western blots of explants cultured without growth factor (control) or with EGF ( iA ) from 5 minutes up to 24 hours, assayed for phosphorylated Akt ( top

    Techniques Used: Western Blot, Cell Culture, IA

    ( iA ) Representative Western blots of explants cultured without growth factor ( lanes 1 and 2 ), with EGF ( lanes 3 and 4 ), or with FGF ( lanes 5 and 6 ) in the presence ( lanes 2 , 4 , and 6 ) or absence ( lanes 1 , 3 , and 5 ) of 50 nM PD153035, added 2 hours before
    Figure Legend Snippet: ( iA ) Representative Western blots of explants cultured without growth factor ( lanes 1 and 2 ), with EGF ( lanes 3 and 4 ), or with FGF ( lanes 5 and 6 ) in the presence ( lanes 2 , 4 , and 6 ) or absence ( lanes 1 , 3 , and 5 ) of 50 nM PD153035, added 2 hours before

    Techniques Used: IA, Western Blot, Cell Culture

    38) Product Images from "Human trophoblast survival at low oxygen concentrations requires metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor"

    Article Title: Human trophoblast survival at low oxygen concentrations requires metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor

    Journal:

    doi: 10.1242/dev.02237

    Regulation of EGF family expression by O 2 . ( A ) Immunohistochemical labeling of EGF family members in cytotrophoblast cells. Antibodies against EGF, TGFα (TGF-a), HBEGF, amphiregulin (AR), betacellulin (BC) and epiregulin (ER) were used, as well
    Figure Legend Snippet: Regulation of EGF family expression by O 2 . ( A ) Immunohistochemical labeling of EGF family members in cytotrophoblast cells. Antibodies against EGF, TGFα (TGF-a), HBEGF, amphiregulin (AR), betacellulin (BC) and epiregulin (ER) were used, as well

    Techniques Used: Expressing, Immunohistochemistry, Labeling

    39) Product Images from "Apical Epidermal Growth Factor Receptor Signaling: Regulation of Stretch-dependent Exocytosis in Bladder Umbrella Cells"

    Article Title: Apical Epidermal Growth Factor Receptor Signaling: Regulation of Stretch-dependent Exocytosis in Bladder Umbrella Cells

    Journal:

    doi: 10.1091/mbc.E06-09-0842

    Expression and distribution of ErbB family receptors in the uroepithelium. (A and B) Total RNA was prepared from rabbit uroepithelium and RT-PCR used to assess expression of ErbB1–4 (A) or EGFR ligands EGF, HB-EGF, and TGFα (B). (C) Localization
    Figure Legend Snippet: Expression and distribution of ErbB family receptors in the uroepithelium. (A and B) Total RNA was prepared from rabbit uroepithelium and RT-PCR used to assess expression of ErbB1–4 (A) or EGFR ligands EGF, HB-EGF, and TGFα (B). (C) Localization

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    40) Product Images from "A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration"

    Article Title: A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration

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

    doi: 10.1073/pnas.0808387106

    lt-hESNSCs remain responsive to instructive regionalization cues. Schematic representation of the experimental protocol ( A ). Compared with control cells treated with FGF2/EGF/B27low ( B ), cells treated with Shh/FGF8 show prominent nuclear immunoreactivity
    Figure Legend Snippet: lt-hESNSCs remain responsive to instructive regionalization cues. Schematic representation of the experimental protocol ( A ). Compared with control cells treated with FGF2/EGF/B27low ( B ), cells treated with Shh/FGF8 show prominent nuclear immunoreactivity

    Techniques Used:

    Related Articles

    Modification:

    Article Title: Cross Talk between c-Met and Epidermal Growth Factor Receptor during Retinal Pigment Epithelial Wound Healing
    Article Snippet: .. The following materials were used: Dulbecco modified essential medium (DMEM), penicillin/streptomycin, and trypsin (Invitrogen, Carlsbad, CA); human recombinant HGF, HB-EGF, and EGF (R & D Systems, Minneapolis, MN); GM6001, a hydroxamic acid matrix metalloproteinase (MMP) inhibitor (3-( N -hydroxycarbamoyl)-2-( R )-isobutylpropionyl- l -tryptophan methylamide; Calbiochem, La Jolla, CA); antibodies against human EGFR (erbB1), erbB4, ERK 2 (p42 MAPK), phosphorylated ERK1/2 (p44/p42 MAPK), PY99, and Met (c-28; Santa Cruz Biotechnology, Santa Cruz, CA); antibodies against a major substrate of PI3K, AKT, and phospho-AKT (Cell Signaling, Beverly, MA); rabbit anti-EGFR (Tyr 845; Biosource, Camarillo, CA); c-Met antibody that recognizes the extracellular region of Met (Upstate Biotechnology, Lake Placid, NY); antibodies against erbB2 and erbB3 (Laboratory Vision; Fremont, CA); Boyden chamber (48 wells; Neuroprobe, Cabin John, MD) and polycarbonate membranes (14- μ m pores; Osmonics, Inc., Livermore, CA); hydroxyurea, tyrphostin AG 1478, and all other chemicals (Sigma-Aldrich, St. Louis, MO). .. ARPE-19, the cell line most frequently used to study RPE function in vitro, was purchased (American Type Culture Collection, Manassas, VA).

    Incubation:

    Article Title: Hemangiosarcoma and its Cancer Stem Cell Sub-Population are Effectively Killed by a Toxin Targeted through Epidermal Growth Factor and Urokinase Receptors
    Article Snippet: .. For antibody blocking assays, cells were incubated in 96 well plates for 24 hours preceding the addition of 0.1 nM EGFuPA-toxin and varying concentrations of anti-human EGF antibody (R & D Systems, Minneapolis, MN) or varying concentrations of anti-uPA antibody (American Diagnostica Inc., Stamford, CT), as indicated. .. The mouse leukocyte specific antibody anti-Ly5.2 was included at the same concentrations as a negative control .

    Recombinant:

    Article Title: Epidermal Growth Factor (EGF) Induces Corneal Keratocyte Differentiation via PI-3 Kinase Activity. Synergism with TGF-β1
    Article Snippet: .. Recombinant human EGF, TGF-β and goat anti-EGF antibody (AB-236-NA) were purchased from R & D Systems, Inc. (Minneapolis, MN); 4-(3-chloroanilino)-6, 7-dimethoxyquinazoline (AG1478) from Biosource International, Inc. (Camarillo, CA). .. 4, 6-diamidino-2-phenylindole (DAPI), mouse monoclonal α-SMA, anti-vimentin (clone Vim-13.2), anti-chondroitin sulfate (clone CS-56), anti-collagen type IV (clone Col-94), anti-cellular fibronectin (clone FN-3E2), anti-human fibronectin (clone IST-3) and antilaminin (clone LAM-89), anti-proliferating cell protein Ki-67(clone pp-67) were purchased from Sigma (St. Louis, MO).

    Article Title: Renal Fibrosis
    Article Snippet: .. Recombinant human TGF-β1 , human epithelial growth factor (EGF), and the neutralizing polyclonal goat antibodies to TGF-β and EGF were purchased from R & D Systems (Minneapolis, MN). .. Mouse monoclonal antibody to vimentin was obtained from Boehringer Mannheim (Mannheim, Germany).

    Article Title: Cross Talk between c-Met and Epidermal Growth Factor Receptor during Retinal Pigment Epithelial Wound Healing
    Article Snippet: .. The following materials were used: Dulbecco modified essential medium (DMEM), penicillin/streptomycin, and trypsin (Invitrogen, Carlsbad, CA); human recombinant HGF, HB-EGF, and EGF (R & D Systems, Minneapolis, MN); GM6001, a hydroxamic acid matrix metalloproteinase (MMP) inhibitor (3-( N -hydroxycarbamoyl)-2-( R )-isobutylpropionyl- l -tryptophan methylamide; Calbiochem, La Jolla, CA); antibodies against human EGFR (erbB1), erbB4, ERK 2 (p42 MAPK), phosphorylated ERK1/2 (p44/p42 MAPK), PY99, and Met (c-28; Santa Cruz Biotechnology, Santa Cruz, CA); antibodies against a major substrate of PI3K, AKT, and phospho-AKT (Cell Signaling, Beverly, MA); rabbit anti-EGFR (Tyr 845; Biosource, Camarillo, CA); c-Met antibody that recognizes the extracellular region of Met (Upstate Biotechnology, Lake Placid, NY); antibodies against erbB2 and erbB3 (Laboratory Vision; Fremont, CA); Boyden chamber (48 wells; Neuroprobe, Cabin John, MD) and polycarbonate membranes (14- μ m pores; Osmonics, Inc., Livermore, CA); hydroxyurea, tyrphostin AG 1478, and all other chemicals (Sigma-Aldrich, St. Louis, MO). .. ARPE-19, the cell line most frequently used to study RPE function in vitro, was purchased (American Type Culture Collection, Manassas, VA).

    other:

    Article Title: Quantitative analysis of the responses of murine bone marrow mesenchymal stem cells to EGF, PDGF-BB and fibronectin by factorial design methodology
    Article Snippet: A two-level full factorial experimental design was applied to investigate the influence of EGF (R & D Systems), PDGF-BB (Chemicon) and FN (Chemicon), as well as their interactions on cell proliferation, cell morphology and cell cycle profile of BMSCs.

    Blocking Assay:

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    Article Snippet: .. Caco-2 cells were cocultured in the presence of EGC or CCD18Co myofibroblasts seeded on the bottom of 6- or 12-well plates or in the presence of either EGC-CM, PP2 (Calbiochem), GM6001 (Millipore), PD153035 (Calbiochem), EGFR blocking antibody (Calbiochem), EGF blocking antibody (R & D Systems), hEGF (Sigma), rEGF (R & D Systems), or hproEGF (R & D Systems). .. Caco-2 monolayers were wounded by using a tip attached to a 0.5- to 10-μl pipette.

    Article Title: Hemangiosarcoma and its Cancer Stem Cell Sub-Population are Effectively Killed by a Toxin Targeted through Epidermal Growth Factor and Urokinase Receptors
    Article Snippet: .. For antibody blocking assays, cells were incubated in 96 well plates for 24 hours preceding the addition of 0.1 nM EGFuPA-toxin and varying concentrations of anti-human EGF antibody (R & D Systems, Minneapolis, MN) or varying concentrations of anti-uPA antibody (American Diagnostica Inc., Stamford, CT), as indicated. .. The mouse leukocyte specific antibody anti-Ly5.2 was included at the same concentrations as a negative control .

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    R&D Systems egf detection
    Targeting CAFs in ascites disrupts MUs and attenuates OC dissemination. (A) Cell viability analysis of the OC tumor cells and of primary CAFs (#1–3) exposed for 48 h to varying doses of imatinib (ima; 0–100 nM). (B) Representative images and quantification of spheroid formation by primary CAFs (#2, 3, and 5) or in suspended coculture with GFP-transfected tumor cells, in the presence or absence of 20 nM imatinib. Bar, 50 µm. (C) Flow cytometry analysis of cellular apoptosis rate for GFP + SKOV3 cells cultured in heterotypic spheroids with CAFs (#2, 5, and 6) for the indicated time, in the presence or absence of 20 nM imatinib. (D) <t>EGF</t> secretion by CAFs (#3, 5, and 8) cocultured or not with SKOV3 cells, in the presence or absence of varying doses of imatinib, was assessed by <t>ELISA.</t> (E) Immunoblot of ITGA5 in control SKOV3 and OV90 cells, or after heterotypic coculture with CAFs (#5 and 8), in the presence or absence of 20 nM imatinib. (F) Representative images of peritoneal sphere adhesion 1 wk after coimplantation of SKOV3 cells with ima-primed CAFs (#3, 7, and 8) or untreated controls. Bar, 50 µm. (G–I) Representative bioluminescence images (G), tumor growth curves (H), and survival curves (I) in SKOV3-Luc tumor-bearing mice coimplanted with imatinib-primed CAFs (#7 and 8) or untreated controls ( n = 10 mice per group). (J) H E and Masson’s trichrome staining of tumors from mice implanted with SKOV3-Luc cells only or coimplanted with ima-primed or untreated CAFs. Bars, 50 µm (left) and 100 µm (right). Data are means ± SEM and representative of four (A–C), two (D, E, and G–J) or three (F) independent experiments. *, P
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    Serum markers of inflammation, proliferation, and angiogenesis. C-reactive protein (a), <t>interleukin-1</t> β (b), interleukin-2 (c), vascular endothelial growth factor (d), transforming growth factor beta-1 (e), and interleukin-7 (f). Values are mean ± SEM. * P
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    Targeting CAFs in ascites disrupts MUs and attenuates OC dissemination. (A) Cell viability analysis of the OC tumor cells and of primary CAFs (#1–3) exposed for 48 h to varying doses of imatinib (ima; 0–100 nM). (B) Representative images and quantification of spheroid formation by primary CAFs (#2, 3, and 5) or in suspended coculture with GFP-transfected tumor cells, in the presence or absence of 20 nM imatinib. Bar, 50 µm. (C) Flow cytometry analysis of cellular apoptosis rate for GFP + SKOV3 cells cultured in heterotypic spheroids with CAFs (#2, 5, and 6) for the indicated time, in the presence or absence of 20 nM imatinib. (D) <t>EGF</t> secretion by CAFs (#3, 5, and 8) cocultured or not with SKOV3 cells, in the presence or absence of varying doses of imatinib, was assessed by <t>ELISA.</t> (E) Immunoblot of ITGA5 in control SKOV3 and OV90 cells, or after heterotypic coculture with CAFs (#5 and 8), in the presence or absence of 20 nM imatinib. (F) Representative images of peritoneal sphere adhesion 1 wk after coimplantation of SKOV3 cells with ima-primed CAFs (#3, 7, and 8) or untreated controls. Bar, 50 µm. (G–I) Representative bioluminescence images (G), tumor growth curves (H), and survival curves (I) in SKOV3-Luc tumor-bearing mice coimplanted with imatinib-primed CAFs (#7 and 8) or untreated controls ( n = 10 mice per group). (J) H E and Masson’s trichrome staining of tumors from mice implanted with SKOV3-Luc cells only or coimplanted with ima-primed or untreated CAFs. Bars, 50 µm (left) and 100 µm (right). Data are means ± SEM and representative of four (A–C), two (D, E, and G–J) or three (F) independent experiments. *, P
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    Targeting CAFs in ascites disrupts MUs and attenuates OC dissemination. (A) Cell viability analysis of the OC tumor cells and of primary CAFs (#1–3) exposed for 48 h to varying doses of imatinib (ima; 0–100 nM). (B) Representative images and quantification of spheroid formation by primary CAFs (#2, 3, and 5) or in suspended coculture with GFP-transfected tumor cells, in the presence or absence of 20 nM imatinib. Bar, 50 µm. (C) Flow cytometry analysis of cellular apoptosis rate for GFP + SKOV3 cells cultured in heterotypic spheroids with CAFs (#2, 5, and 6) for the indicated time, in the presence or absence of 20 nM imatinib. (D) EGF secretion by CAFs (#3, 5, and 8) cocultured or not with SKOV3 cells, in the presence or absence of varying doses of imatinib, was assessed by ELISA. (E) Immunoblot of ITGA5 in control SKOV3 and OV90 cells, or after heterotypic coculture with CAFs (#5 and 8), in the presence or absence of 20 nM imatinib. (F) Representative images of peritoneal sphere adhesion 1 wk after coimplantation of SKOV3 cells with ima-primed CAFs (#3, 7, and 8) or untreated controls. Bar, 50 µm. (G–I) Representative bioluminescence images (G), tumor growth curves (H), and survival curves (I) in SKOV3-Luc tumor-bearing mice coimplanted with imatinib-primed CAFs (#7 and 8) or untreated controls ( n = 10 mice per group). (J) H E and Masson’s trichrome staining of tumors from mice implanted with SKOV3-Luc cells only or coimplanted with ima-primed or untreated CAFs. Bars, 50 µm (left) and 100 µm (right). Data are means ± SEM and representative of four (A–C), two (D, E, and G–J) or three (F) independent experiments. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Heterotypic CAF-tumor spheroids promote early peritoneal metastatis of ovarian cancer

    doi: 10.1084/jem.20180765

    Figure Lengend Snippet: Targeting CAFs in ascites disrupts MUs and attenuates OC dissemination. (A) Cell viability analysis of the OC tumor cells and of primary CAFs (#1–3) exposed for 48 h to varying doses of imatinib (ima; 0–100 nM). (B) Representative images and quantification of spheroid formation by primary CAFs (#2, 3, and 5) or in suspended coculture with GFP-transfected tumor cells, in the presence or absence of 20 nM imatinib. Bar, 50 µm. (C) Flow cytometry analysis of cellular apoptosis rate for GFP + SKOV3 cells cultured in heterotypic spheroids with CAFs (#2, 5, and 6) for the indicated time, in the presence or absence of 20 nM imatinib. (D) EGF secretion by CAFs (#3, 5, and 8) cocultured or not with SKOV3 cells, in the presence or absence of varying doses of imatinib, was assessed by ELISA. (E) Immunoblot of ITGA5 in control SKOV3 and OV90 cells, or after heterotypic coculture with CAFs (#5 and 8), in the presence or absence of 20 nM imatinib. (F) Representative images of peritoneal sphere adhesion 1 wk after coimplantation of SKOV3 cells with ima-primed CAFs (#3, 7, and 8) or untreated controls. Bar, 50 µm. (G–I) Representative bioluminescence images (G), tumor growth curves (H), and survival curves (I) in SKOV3-Luc tumor-bearing mice coimplanted with imatinib-primed CAFs (#7 and 8) or untreated controls ( n = 10 mice per group). (J) H E and Masson’s trichrome staining of tumors from mice implanted with SKOV3-Luc cells only or coimplanted with ima-primed or untreated CAFs. Bars, 50 µm (left) and 100 µm (right). Data are means ± SEM and representative of four (A–C), two (D, E, and G–J) or three (F) independent experiments. *, P

    Article Snippet: EGF detection by ELISA EGF protein levels in patient malignant ascites and CM of CAFs were measured by ELISA kits (R & D Systems) according to the manufacturer’s protocol.

    Techniques: Transfection, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Mouse Assay, Staining

    CAF-dependent EGF secretion is required for ATC ITGA5 expression in MUs. (A) Cytokine profile from control CAFs (#1), and from CAFs primed either with SKOV3 CM or with TGF-β1 (100 ng ml –1 ) for 48 h. The shared cytokines elevated in CAFs treated with CM or TGF-β1 are listed. (B) Western blot analysis of ITGA5 in SKOV3 cells treated with variant cytokines for 48 h. (C) Representative immunofluorescence images of EGF in SKOV3 and CAFs (#8 and 9) in adherent culture, in SKOV3 homospheroids, and in MUs formed by SKOV3 and CAFs. Bar, 50 µm. (D) EGF secretion in the MU microenvironment and in the corresponding ascites macroenvironment of eight HGSOC patients was assessed by ELISA. (E) Immunoblotting for ITGA5 in control and in isolated tumor cells following coculture with CAFs (#6 and 8) in MUs, in the presence or absence of either EGF-neutralizing antibody or IgG control. (F) Schematic representation of the ITGA5 promoter reporter constructs (top) and analysis of ITGA5 promoter activity (bottom) in variant tumor cell groups, as described in E. (G) Representative images and quantification of heterotypic spheroid formation by SKOV3 and CAFs (#4, 9, and 10), in the presence or absence of IgG or EGF-neutralizing antibody. Bar, 100 µm. (H and I) Representative i.p. bioluminescence images (H) and survival curves (I) for mice bearing tumors generated by coinjection of SKOV3-Luc and CAFs (#7 and 9) in the control IgG and the EGF-neutralizing antibody group ( n = 10 mice per group). (J) Immunoblot of ITGA5 in peritoneal tumor cells isolated from the groups treated with either control IgG or EGF-neutralizing antibody. (K) Editing EGFR in SKOV3 using the CRISPR/Cas9 system. Top: Schematic diagram of sgRNA1-3 targeting exon 3/5 of the EGFR gene. Bottom: The genomic PCR products of SKOV3 cells transduced with scrambled sgRNA (mock) or sgRNA1-3 were analyzed with T7E1 assay. (L) SKOV3 cells transduced with scrambled sgRNA (control) or sgRNA1-3 were subjected to immunoblotting with EGFR. (M) Tumor growth curves developed by control and EGFR-deficient SKOV3-Luc cells in combination with CAFs (#9 and 10; n = 10 mice per group). Data are means ± SEM and representative of two (A, B, E, and H–M) or three (C, D, F, and G) independent experiments. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Heterotypic CAF-tumor spheroids promote early peritoneal metastatis of ovarian cancer

    doi: 10.1084/jem.20180765

    Figure Lengend Snippet: CAF-dependent EGF secretion is required for ATC ITGA5 expression in MUs. (A) Cytokine profile from control CAFs (#1), and from CAFs primed either with SKOV3 CM or with TGF-β1 (100 ng ml –1 ) for 48 h. The shared cytokines elevated in CAFs treated with CM or TGF-β1 are listed. (B) Western blot analysis of ITGA5 in SKOV3 cells treated with variant cytokines for 48 h. (C) Representative immunofluorescence images of EGF in SKOV3 and CAFs (#8 and 9) in adherent culture, in SKOV3 homospheroids, and in MUs formed by SKOV3 and CAFs. Bar, 50 µm. (D) EGF secretion in the MU microenvironment and in the corresponding ascites macroenvironment of eight HGSOC patients was assessed by ELISA. (E) Immunoblotting for ITGA5 in control and in isolated tumor cells following coculture with CAFs (#6 and 8) in MUs, in the presence or absence of either EGF-neutralizing antibody or IgG control. (F) Schematic representation of the ITGA5 promoter reporter constructs (top) and analysis of ITGA5 promoter activity (bottom) in variant tumor cell groups, as described in E. (G) Representative images and quantification of heterotypic spheroid formation by SKOV3 and CAFs (#4, 9, and 10), in the presence or absence of IgG or EGF-neutralizing antibody. Bar, 100 µm. (H and I) Representative i.p. bioluminescence images (H) and survival curves (I) for mice bearing tumors generated by coinjection of SKOV3-Luc and CAFs (#7 and 9) in the control IgG and the EGF-neutralizing antibody group ( n = 10 mice per group). (J) Immunoblot of ITGA5 in peritoneal tumor cells isolated from the groups treated with either control IgG or EGF-neutralizing antibody. (K) Editing EGFR in SKOV3 using the CRISPR/Cas9 system. Top: Schematic diagram of sgRNA1-3 targeting exon 3/5 of the EGFR gene. Bottom: The genomic PCR products of SKOV3 cells transduced with scrambled sgRNA (mock) or sgRNA1-3 were analyzed with T7E1 assay. (L) SKOV3 cells transduced with scrambled sgRNA (control) or sgRNA1-3 were subjected to immunoblotting with EGFR. (M) Tumor growth curves developed by control and EGFR-deficient SKOV3-Luc cells in combination with CAFs (#9 and 10; n = 10 mice per group). Data are means ± SEM and representative of two (A, B, E, and H–M) or three (C, D, F, and G) independent experiments. *, P

    Article Snippet: EGF detection by ELISA EGF protein levels in patient malignant ascites and CM of CAFs were measured by ELISA kits (R & D Systems) according to the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Variant Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Isolation, Construct, Activity Assay, Mouse Assay, Generated, CRISPR, Polymerase Chain Reaction, Transduction

    Serum markers of inflammation, proliferation, and angiogenesis. C-reactive protein (a), interleukin-1 β (b), interleukin-2 (c), vascular endothelial growth factor (d), transforming growth factor beta-1 (e), and interleukin-7 (f). Values are mean ± SEM. * P

    Journal: BioMed Research International

    Article Title: Conversion to Sirolimus Ameliorates Cyclosporine-Induced Nephropathy in the Rat: Focus on Serum, Urine, Gene, and Protein Renal Expression Biomarkers

    doi: 10.1155/2014/576929

    Figure Lengend Snippet: Serum markers of inflammation, proliferation, and angiogenesis. C-reactive protein (a), interleukin-1 β (b), interleukin-2 (c), vascular endothelial growth factor (d), transforming growth factor beta-1 (e), and interleukin-7 (f). Values are mean ± SEM. * P

    Article Snippet: Serum levels of interleukin 1β (IL-1β ), interleukin 2 (IL-2), vascular epidermal growth factor (VEGF), and transforming growth factor beta 1 (TGF-β 1 ) were measured by ultrasensitive Quantikine ELISA kits (R & D Systems, Minneapolis, USA).

    Techniques:

    Kidney mRNA expression of proliferation, inflammation, and angiogenesis mediators. PCNA (a), TP53 (b), mTOR (c), TGF- β 1 (d), NF- κ B (e), and Mki67 (f) as proliferation status markers; CRP (g), TNF- α (h), IL-2 (i), COX-2 (j), and IL-1 β (k) as inflammation status markers and VEGF (l) as angiogenesis status marker. Values are mean of CNRQ (calibrated normalized relative quantities) of the control ± SEM. * P

    Journal: BioMed Research International

    Article Title: Conversion to Sirolimus Ameliorates Cyclosporine-Induced Nephropathy in the Rat: Focus on Serum, Urine, Gene, and Protein Renal Expression Biomarkers

    doi: 10.1155/2014/576929

    Figure Lengend Snippet: Kidney mRNA expression of proliferation, inflammation, and angiogenesis mediators. PCNA (a), TP53 (b), mTOR (c), TGF- β 1 (d), NF- κ B (e), and Mki67 (f) as proliferation status markers; CRP (g), TNF- α (h), IL-2 (i), COX-2 (j), and IL-1 β (k) as inflammation status markers and VEGF (l) as angiogenesis status marker. Values are mean of CNRQ (calibrated normalized relative quantities) of the control ± SEM. * P

    Article Snippet: Serum levels of interleukin 1β (IL-1β ), interleukin 2 (IL-2), vascular epidermal growth factor (VEGF), and transforming growth factor beta 1 (TGF-β 1 ) were measured by ultrasensitive Quantikine ELISA kits (R & D Systems, Minneapolis, USA).

    Techniques: Expressing, Marker

    Targeting CAFs in ascites disrupts MUs and attenuates OC dissemination. (A) Cell viability analysis of the OC tumor cells and of primary CAFs (#1–3) exposed for 48 h to varying doses of imatinib (ima; 0–100 nM). (B) Representative images and quantification of spheroid formation by primary CAFs (#2, 3, and 5) or in suspended coculture with GFP-transfected tumor cells, in the presence or absence of 20 nM imatinib. Bar, 50 µm. (C) Flow cytometry analysis of cellular apoptosis rate for GFP + SKOV3 cells cultured in heterotypic spheroids with CAFs (#2, 5, and 6) for the indicated time, in the presence or absence of 20 nM imatinib. (D) EGF secretion by CAFs (#3, 5, and 8) cocultured or not with SKOV3 cells, in the presence or absence of varying doses of imatinib, was assessed by ELISA. (E) Immunoblot of ITGA5 in control SKOV3 and OV90 cells, or after heterotypic coculture with CAFs (#5 and 8), in the presence or absence of 20 nM imatinib. (F) Representative images of peritoneal sphere adhesion 1 wk after coimplantation of SKOV3 cells with ima-primed CAFs (#3, 7, and 8) or untreated controls. Bar, 50 µm. (G–I) Representative bioluminescence images (G), tumor growth curves (H), and survival curves (I) in SKOV3-Luc tumor-bearing mice coimplanted with imatinib-primed CAFs (#7 and 8) or untreated controls ( n = 10 mice per group). (J) H E and Masson’s trichrome staining of tumors from mice implanted with SKOV3-Luc cells only or coimplanted with ima-primed or untreated CAFs. Bars, 50 µm (left) and 100 µm (right). Data are means ± SEM and representative of four (A–C), two (D, E, and G–J) or three (F) independent experiments. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Heterotypic CAF-tumor spheroids promote early peritoneal metastatis of ovarian cancer

    doi: 10.1084/jem.20180765

    Figure Lengend Snippet: Targeting CAFs in ascites disrupts MUs and attenuates OC dissemination. (A) Cell viability analysis of the OC tumor cells and of primary CAFs (#1–3) exposed for 48 h to varying doses of imatinib (ima; 0–100 nM). (B) Representative images and quantification of spheroid formation by primary CAFs (#2, 3, and 5) or in suspended coculture with GFP-transfected tumor cells, in the presence or absence of 20 nM imatinib. Bar, 50 µm. (C) Flow cytometry analysis of cellular apoptosis rate for GFP + SKOV3 cells cultured in heterotypic spheroids with CAFs (#2, 5, and 6) for the indicated time, in the presence or absence of 20 nM imatinib. (D) EGF secretion by CAFs (#3, 5, and 8) cocultured or not with SKOV3 cells, in the presence or absence of varying doses of imatinib, was assessed by ELISA. (E) Immunoblot of ITGA5 in control SKOV3 and OV90 cells, or after heterotypic coculture with CAFs (#5 and 8), in the presence or absence of 20 nM imatinib. (F) Representative images of peritoneal sphere adhesion 1 wk after coimplantation of SKOV3 cells with ima-primed CAFs (#3, 7, and 8) or untreated controls. Bar, 50 µm. (G–I) Representative bioluminescence images (G), tumor growth curves (H), and survival curves (I) in SKOV3-Luc tumor-bearing mice coimplanted with imatinib-primed CAFs (#7 and 8) or untreated controls ( n = 10 mice per group). (J) H E and Masson’s trichrome staining of tumors from mice implanted with SKOV3-Luc cells only or coimplanted with ima-primed or untreated CAFs. Bars, 50 µm (left) and 100 µm (right). Data are means ± SEM and representative of four (A–C), two (D, E, and G–J) or three (F) independent experiments. *, P

    Article Snippet: EGF detection by ELISA EGF protein levels in patient malignant ascites and CM of CAFs were measured by ELISA kits (R & D Systems) according to the manufacturer’s protocol.

    Techniques: Transfection, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Mouse Assay, Staining

    CAF-dependent EGF secretion is required for ATC ITGA5 expression in MUs. (A) Cytokine profile from control CAFs (#1), and from CAFs primed either with SKOV3 CM or with TGF-β1 (100 ng ml –1 ) for 48 h. The shared cytokines elevated in CAFs treated with CM or TGF-β1 are listed. (B) Western blot analysis of ITGA5 in SKOV3 cells treated with variant cytokines for 48 h. (C) Representative immunofluorescence images of EGF in SKOV3 and CAFs (#8 and 9) in adherent culture, in SKOV3 homospheroids, and in MUs formed by SKOV3 and CAFs. Bar, 50 µm. (D) EGF secretion in the MU microenvironment and in the corresponding ascites macroenvironment of eight HGSOC patients was assessed by ELISA. (E) Immunoblotting for ITGA5 in control and in isolated tumor cells following coculture with CAFs (#6 and 8) in MUs, in the presence or absence of either EGF-neutralizing antibody or IgG control. (F) Schematic representation of the ITGA5 promoter reporter constructs (top) and analysis of ITGA5 promoter activity (bottom) in variant tumor cell groups, as described in E. (G) Representative images and quantification of heterotypic spheroid formation by SKOV3 and CAFs (#4, 9, and 10), in the presence or absence of IgG or EGF-neutralizing antibody. Bar, 100 µm. (H and I) Representative i.p. bioluminescence images (H) and survival curves (I) for mice bearing tumors generated by coinjection of SKOV3-Luc and CAFs (#7 and 9) in the control IgG and the EGF-neutralizing antibody group ( n = 10 mice per group). (J) Immunoblot of ITGA5 in peritoneal tumor cells isolated from the groups treated with either control IgG or EGF-neutralizing antibody. (K) Editing EGFR in SKOV3 using the CRISPR/Cas9 system. Top: Schematic diagram of sgRNA1-3 targeting exon 3/5 of the EGFR gene. Bottom: The genomic PCR products of SKOV3 cells transduced with scrambled sgRNA (mock) or sgRNA1-3 were analyzed with T7E1 assay. (L) SKOV3 cells transduced with scrambled sgRNA (control) or sgRNA1-3 were subjected to immunoblotting with EGFR. (M) Tumor growth curves developed by control and EGFR-deficient SKOV3-Luc cells in combination with CAFs (#9 and 10; n = 10 mice per group). Data are means ± SEM and representative of two (A, B, E, and H–M) or three (C, D, F, and G) independent experiments. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Heterotypic CAF-tumor spheroids promote early peritoneal metastatis of ovarian cancer

    doi: 10.1084/jem.20180765

    Figure Lengend Snippet: CAF-dependent EGF secretion is required for ATC ITGA5 expression in MUs. (A) Cytokine profile from control CAFs (#1), and from CAFs primed either with SKOV3 CM or with TGF-β1 (100 ng ml –1 ) for 48 h. The shared cytokines elevated in CAFs treated with CM or TGF-β1 are listed. (B) Western blot analysis of ITGA5 in SKOV3 cells treated with variant cytokines for 48 h. (C) Representative immunofluorescence images of EGF in SKOV3 and CAFs (#8 and 9) in adherent culture, in SKOV3 homospheroids, and in MUs formed by SKOV3 and CAFs. Bar, 50 µm. (D) EGF secretion in the MU microenvironment and in the corresponding ascites macroenvironment of eight HGSOC patients was assessed by ELISA. (E) Immunoblotting for ITGA5 in control and in isolated tumor cells following coculture with CAFs (#6 and 8) in MUs, in the presence or absence of either EGF-neutralizing antibody or IgG control. (F) Schematic representation of the ITGA5 promoter reporter constructs (top) and analysis of ITGA5 promoter activity (bottom) in variant tumor cell groups, as described in E. (G) Representative images and quantification of heterotypic spheroid formation by SKOV3 and CAFs (#4, 9, and 10), in the presence or absence of IgG or EGF-neutralizing antibody. Bar, 100 µm. (H and I) Representative i.p. bioluminescence images (H) and survival curves (I) for mice bearing tumors generated by coinjection of SKOV3-Luc and CAFs (#7 and 9) in the control IgG and the EGF-neutralizing antibody group ( n = 10 mice per group). (J) Immunoblot of ITGA5 in peritoneal tumor cells isolated from the groups treated with either control IgG or EGF-neutralizing antibody. (K) Editing EGFR in SKOV3 using the CRISPR/Cas9 system. Top: Schematic diagram of sgRNA1-3 targeting exon 3/5 of the EGFR gene. Bottom: The genomic PCR products of SKOV3 cells transduced with scrambled sgRNA (mock) or sgRNA1-3 were analyzed with T7E1 assay. (L) SKOV3 cells transduced with scrambled sgRNA (control) or sgRNA1-3 were subjected to immunoblotting with EGFR. (M) Tumor growth curves developed by control and EGFR-deficient SKOV3-Luc cells in combination with CAFs (#9 and 10; n = 10 mice per group). Data are means ± SEM and representative of two (A, B, E, and H–M) or three (C, D, F, and G) independent experiments. *, P

    Article Snippet: EGF detection by ELISA EGF protein levels in patient malignant ascites and CM of CAFs were measured by ELISA kits (R & D Systems) according to the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Variant Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Isolation, Construct, Activity Assay, Mouse Assay, Generated, CRISPR, Polymerase Chain Reaction, Transduction

    Transcriptional activation of EGF gene by S100A11. (A) Transcription factors bound to the EGF promoter identified by a newly developed method. NHK were precultured in HKGS-free EpiLife for 24 h. An Akt inhibitor and anti-RAGE antibody were added 1 h before

    Journal: Molecular Biology of the Cell

    Article Title: S100A11, an Dual Mediator for Growth Regulation of Human Keratinocytes

    doi: 10.1091/mbc.E07-07-0682

    Figure Lengend Snippet: Transcriptional activation of EGF gene by S100A11. (A) Transcription factors bound to the EGF promoter identified by a newly developed method. NHK were precultured in HKGS-free EpiLife for 24 h. An Akt inhibitor and anti-RAGE antibody were added 1 h before

    Article Snippet: Recombinant proteins of human IL-1F9, IL-8/CXCL8, CXCL1, and soluble receptor for advanced glycation end products (RAGE) (sRAGE/Fc chimera), neutralizing mouse antibodies against human EGF and RAGE, and control mouse immunoglobulin (Ig)G (R & D Systems, Minneapolis, MN) were obtained from the designated sources.

    Techniques: Activation Assay