Structured Review

PeproTech human recombinant egf
GDF15 promotes the response to <t>EGF</t> and EGFR expression in hippocampal precursors. (A-D) Quantitative analysis of the total cell number (A,C,D) and clones (B) obtained upon plating E18 (A-C) and E14 (D) wild-type and Gdf15 -/- cells in both EGF (E) and <t>FGF2</t> (F) (A,B,D), or E and/or F, as indicated (C). In A, exogenous GDF15 was added as shown. In C, values are the percentage variation of the total cell number scored in parallel wild-type cultures grown in both growth factors. (E,F) Representative dot plots of dissociated E18 wild-type (E) and Gdf15 -/- (F) HP after staining with EGF-Alexa 488 and PI to reveal EGFR low (R1), EGFR high (R2) and dead cells (R3). Values represent the means±s.e.m. of the percentage of total cells in each gate. (G,H) Quantitative analysis of the EGFR high cell numbers in the E18 wild-type and Gdf15 -/- HP measured at day in vitro (DIV) 0, or after 24 hours exposure to FGF2 (DIV 1) (G) and of the percentage of E18 wild-type and Gdf15 -/- EGFR high cells capable of undergoing clone formation upon plating at DIV0 (H). * and # indicate values that are significantly different from the corresponding wild-type samples and the counterpart at DIV0, respectively. * P ≤0.05; ** P ≤0.01; ***,### P ≤0.001; n ≥3.
Human Recombinant Egf, supplied by PeproTech, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/human recombinant egf/product/PeproTech
Average 97 stars, based on 1 article reviews
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human recombinant egf - by Bioz Stars, 2021-03
97/100 stars

Images

1) Product Images from "Growth/differentiation factor 15 promotes EGFR signalling, and regulates proliferation and migration in the hippocampus of neonatal and young adult mice"

Article Title: Growth/differentiation factor 15 promotes EGFR signalling, and regulates proliferation and migration in the hippocampus of neonatal and young adult mice

Journal: Development (Cambridge, England)

doi: 10.1242/dev.096131

GDF15 promotes the response to EGF and EGFR expression in hippocampal precursors. (A-D) Quantitative analysis of the total cell number (A,C,D) and clones (B) obtained upon plating E18 (A-C) and E14 (D) wild-type and Gdf15 -/- cells in both EGF (E) and FGF2 (F) (A,B,D), or E and/or F, as indicated (C). In A, exogenous GDF15 was added as shown. In C, values are the percentage variation of the total cell number scored in parallel wild-type cultures grown in both growth factors. (E,F) Representative dot plots of dissociated E18 wild-type (E) and Gdf15 -/- (F) HP after staining with EGF-Alexa 488 and PI to reveal EGFR low (R1), EGFR high (R2) and dead cells (R3). Values represent the means±s.e.m. of the percentage of total cells in each gate. (G,H) Quantitative analysis of the EGFR high cell numbers in the E18 wild-type and Gdf15 -/- HP measured at day in vitro (DIV) 0, or after 24 hours exposure to FGF2 (DIV 1) (G) and of the percentage of E18 wild-type and Gdf15 -/- EGFR high cells capable of undergoing clone formation upon plating at DIV0 (H). * and # indicate values that are significantly different from the corresponding wild-type samples and the counterpart at DIV0, respectively. * P ≤0.05; ** P ≤0.01; ***,### P ≤0.001; n ≥3.
Figure Legend Snippet: GDF15 promotes the response to EGF and EGFR expression in hippocampal precursors. (A-D) Quantitative analysis of the total cell number (A,C,D) and clones (B) obtained upon plating E18 (A-C) and E14 (D) wild-type and Gdf15 -/- cells in both EGF (E) and FGF2 (F) (A,B,D), or E and/or F, as indicated (C). In A, exogenous GDF15 was added as shown. In C, values are the percentage variation of the total cell number scored in parallel wild-type cultures grown in both growth factors. (E,F) Representative dot plots of dissociated E18 wild-type (E) and Gdf15 -/- (F) HP after staining with EGF-Alexa 488 and PI to reveal EGFR low (R1), EGFR high (R2) and dead cells (R3). Values represent the means±s.e.m. of the percentage of total cells in each gate. (G,H) Quantitative analysis of the EGFR high cell numbers in the E18 wild-type and Gdf15 -/- HP measured at day in vitro (DIV) 0, or after 24 hours exposure to FGF2 (DIV 1) (G) and of the percentage of E18 wild-type and Gdf15 -/- EGFR high cells capable of undergoing clone formation upon plating at DIV0 (H). * and # indicate values that are significantly different from the corresponding wild-type samples and the counterpart at DIV0, respectively. * P ≤0.05; ** P ≤0.01; ***,### P ≤0.001; n ≥3.

Techniques Used: Expressing, Staining, In Vitro

2) Product Images from "Tanshinone IIA reverses EGF- and TGF-β1-mediated epithelial-mesenchymal transition in HepG2 cells via the PI3K/Akt/ERK signaling pathway"

Article Title: Tanshinone IIA reverses EGF- and TGF-β1-mediated epithelial-mesenchymal transition in HepG2 cells via the PI3K/Akt/ERK signaling pathway

Journal: Oncology Letters

doi: 10.3892/ol.2019.11032

Effects of Tan IIA on the viability and clonogenic potential of HepG2 cells. (A) Chemical structure of Tan IIA. (B) Cell viability analysis of HepG2 cells cultured in the presence of Tan IIA for 24, 48 and 72 h, by MTT assay. (C) Colony formation assay of HepG2 cells that were untreated or treated with 20 ng/ml EGF, 10 ng/ml TGF-β1 and Tan IIA (0.5, 1 and 2 µM) for 2 weeks. *P
Figure Legend Snippet: Effects of Tan IIA on the viability and clonogenic potential of HepG2 cells. (A) Chemical structure of Tan IIA. (B) Cell viability analysis of HepG2 cells cultured in the presence of Tan IIA for 24, 48 and 72 h, by MTT assay. (C) Colony formation assay of HepG2 cells that were untreated or treated with 20 ng/ml EGF, 10 ng/ml TGF-β1 and Tan IIA (0.5, 1 and 2 µM) for 2 weeks. *P

Techniques Used: Cell Culture, MTT Assay, Colony Assay

Effects of Tan IIA on EGF- and TGF-β1-induced migration and invasion of HepG2 cells. The migration and invasion of HepG2 cells that were untreated or treated with (A) 20 ng/ml EGF, (B) 10 ng/ml TGF-β1 and Tan IIA (0.5, 1 and 2 µM) were analyzed by wound healing and Transwell assays, respectively. Scale bars, 100 µm. **P
Figure Legend Snippet: Effects of Tan IIA on EGF- and TGF-β1-induced migration and invasion of HepG2 cells. The migration and invasion of HepG2 cells that were untreated or treated with (A) 20 ng/ml EGF, (B) 10 ng/ml TGF-β1 and Tan IIA (0.5, 1 and 2 µM) were analyzed by wound healing and Transwell assays, respectively. Scale bars, 100 µm. **P

Techniques Used: Migration

Tan IIA inhibits EMT by deactivating the PI3K/Akt/ERK signaling pathway in EGF- and TGF-β1-treated HepG2 cells. (A) Phosphorylation and expression of Akt and ERK1/2 in HepG2 cells that were untreated or treated with 20 ng/ml EGF, 10 ng/ml TGF-β1 and 2 µM Tan IIA were analyzed by western blotting. (B) Protein expression levels of E-cadherin, vimentin and Snail in HepG2 cells that were untreated or treated with 20 ng/ml EGF, 10 ng/ml TGF-β1, 20 µM LY, and 20 µM U0126 were analyzed by western blotting. **P
Figure Legend Snippet: Tan IIA inhibits EMT by deactivating the PI3K/Akt/ERK signaling pathway in EGF- and TGF-β1-treated HepG2 cells. (A) Phosphorylation and expression of Akt and ERK1/2 in HepG2 cells that were untreated or treated with 20 ng/ml EGF, 10 ng/ml TGF-β1 and 2 µM Tan IIA were analyzed by western blotting. (B) Protein expression levels of E-cadherin, vimentin and Snail in HepG2 cells that were untreated or treated with 20 ng/ml EGF, 10 ng/ml TGF-β1, 20 µM LY, and 20 µM U0126 were analyzed by western blotting. **P

Techniques Used: Expressing, Western Blot

Tan IIA reverses EGF- and TGF-β1-induced EMT in HepG2 cells. (A) E-cadherin and vimentin expression in HepG2 cells was determined by confocal microscopy. Scale bars, 20 µm. (B) Expression of E-cadherin, MMP-2, N-cadherin, vimentin and Snail in HepG2 cells that were untreated or treated with 20 ng/ml EGF, 10 ng/ml TGF-β1 and 2 µM Tan IIA were analyzed via western blotting. **P
Figure Legend Snippet: Tan IIA reverses EGF- and TGF-β1-induced EMT in HepG2 cells. (A) E-cadherin and vimentin expression in HepG2 cells was determined by confocal microscopy. Scale bars, 20 µm. (B) Expression of E-cadherin, MMP-2, N-cadherin, vimentin and Snail in HepG2 cells that were untreated or treated with 20 ng/ml EGF, 10 ng/ml TGF-β1 and 2 µM Tan IIA were analyzed via western blotting. **P

Techniques Used: Expressing, Confocal Microscopy, Western Blot

3) Product Images from "Bio-Imaging of Colorectal Cancer Models Using Near Infrared Labeled Epidermal Growth Factor"

Article Title: Bio-Imaging of Colorectal Cancer Models Using Near Infrared Labeled Epidermal Growth Factor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048803

Synthesis, purification, spectrum, electrophoresis properties and signaling of EGF-NIR. (A) Reaction scheme for the synthesis of EGF-NIR conjugate, the first amino acid asparagine at the amino terminal is indicated as Asn1. (B) Separation of synthesis reaction mixture on gel permeation chromatography and of EGF-NIR sample from gel permeation on (C) anion exchange chromatography; EGF-NIR-full line (800 nm); gradient of NaCl-broken line; unconjugated EGF-dotted line. (D) HPLC separation of EGF-NIR purified from anion exchanger chromatography. Full line represents absorbance at 226 nm and dotted line indicates the gradient. Insert-12% SDS-PAGE analysis of 10 µg of EGF-NIR scanned with Odyssey and unmodified EGF stained with coomassie blue. (E) NIR spectrum of EGF-NIR [excitation (gray line) and emission (black line)]; Insert-IRDye 800CW NHS ester; (F) EGF-NIR induced Erk phosphorylation.
Figure Legend Snippet: Synthesis, purification, spectrum, electrophoresis properties and signaling of EGF-NIR. (A) Reaction scheme for the synthesis of EGF-NIR conjugate, the first amino acid asparagine at the amino terminal is indicated as Asn1. (B) Separation of synthesis reaction mixture on gel permeation chromatography and of EGF-NIR sample from gel permeation on (C) anion exchange chromatography; EGF-NIR-full line (800 nm); gradient of NaCl-broken line; unconjugated EGF-dotted line. (D) HPLC separation of EGF-NIR purified from anion exchanger chromatography. Full line represents absorbance at 226 nm and dotted line indicates the gradient. Insert-12% SDS-PAGE analysis of 10 µg of EGF-NIR scanned with Odyssey and unmodified EGF stained with coomassie blue. (E) NIR spectrum of EGF-NIR [excitation (gray line) and emission (black line)]; Insert-IRDye 800CW NHS ester; (F) EGF-NIR induced Erk phosphorylation.

Techniques Used: Purification, Electrophoresis, GPC Assay, Chromatography, High Performance Liquid Chromatography, SDS Page, Staining

Related Articles

Incubation:

Article Title: Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy
Article Snippet: On the next day, cells were washed twice with serum-free growth medium before they were incubated with the anti-EGFR antibody directed against the extracellular EGFR domain (clone 108) that was conjugated to colloidal 10 nm gold particles as previously described. .. Optimum gold loading into the cells was observed for 30 min of incubation with 80 nM EGF (Peprotech) present alongside the gold-conjugated EGFR antibody. .. Subsequently, cells were fixed by applying a mixture of 2% (w/v) PFA and 2% (v/v) glutaraldehyde in 0.1 M cacodylate buffer for 30 min. After washing twice with 0.1 M cacodylate buffer, cells were treated with 1% tannic acid in cacodylate buffer and prepared for TEM as previously reported.

other:

Article Title: Human pancreatic cancer stem cells are sensitive to dual inhibition of IGF-IR and ErbB receptors
Article Snippet: Insulin-like growth factor (IGF-I) and Epidermal growth factor (EGF) (Peprotech, Rocky Hill, NJ, USA) were dissolved in phosphate-buffered saline containing 0.1% bovine serum albumin (BSA).

Article Title: High Yield of Adult Oligodendrocyte Lineage Cells Obtained from Meningeal Biopsy
Article Snippet: NS Medium Neurobasal medium (Thermo Fisher Scientific), 2% B27 supplement (Thermo Fisher Scientific), 1% N2 supplement (Thermo Fisher Scientific), 2 mM glutamine (Thermo Fisher Scientific), 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific), 20 ng/ml human EGF (PeproTech) and 20 ng/ml FGF2 (PeproTech).

Article Title: miR-29a contributes to breast cancer cells epithelial–mesenchymal transition, migration, and invasion via down-regulating histone H4K20 trimethylation through directly targeting SUV420H2
Article Snippet: 3D semi-solid spheres culture Three thousand single cells were seeded into 24-well Ultra-Low Attachment Microplates (Corning) in serum-free DMEM/F12 (Invitrogen), supplemented with B27 (1:50, Invitrogen), 20 ng/ml EGF (Peprotech), 10 ng/ml bFGF (Invitrogen), 4 μg/ml insulin (Sigma), and 20% methylcellulose (Sigma).

Concentration Assay:

Article Title: Investigation of brain tissue infiltration by medulloblastoma cells in an ex vivo model
Article Snippet: Reagents Antibodies: goat anti-GFAP (ab53554, 1:300), rabbit anti-Calbindin (ab11426, 1:1000), mouse anti-Calbindin (ab82812, 1:1000), donkey anti-rabbit (ab175649, 1: 100) all from Abcam and donkey anti-goat (A11057, 1:500), Vybrant CM-DiI cell labelling solution (V-2288) from Life Technologies, mouse anti-NeuN clone A60 (MAB377, 1 in 100), mouse anti-GFAP (MAB360, 1:500), rabbit anti-collagen type IV (AB756P, 1:500), anti-nuclei clone 3E1.3(MAB4383) from Millipore, rabbit anti-cleaved caspase 3 (5A1E, 1:500) from Cell Signaling Technology, Click-iT EdU Alexa Fluor-647 imaging kit from Molecular Probes. .. HGF and EGF were ordered from Peprotech and used at a final concentration of 50 ng/mL and 30 ng/mL respectively. ..

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    PeproTech recombinant human egf
    Average cumulative migration of keratinocytes on gradients of immobilized <t>SS-EGF,</t> <t>SS-IGF-1,</t> and SS-IGF-1+SS-EGF and TCPS over 7 days. (a) g2 (power law) gradient patterns of SS-EGF (150 μg/ml), SS-IGF-1 patterned on SS-EGF (each 75 μg/ml
    Recombinant Human Egf, supplied by PeproTech, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human egf/product/PeproTech
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human egf - by Bioz Stars, 2021-03
    97/100 stars
      Buy from Supplier

    96
    PeproTech epidermal growth factor egf
    The Myc 3′ WRE is required for mitogen-induced Myc gene expression. The proliferative regions of colonic crypts were isolated from 7-week-old Myc 3′ WRE −/− mice and WT littermates and were not treated (Ctrl.) or treated for 1 or 3 h with recombinant Wnt3A, <t>R-spondin1,</t> and <t>EGF</t> (A), Wnt3A and R-spondin1 (B), or EGF (C). RNAs were isolated, cDNAs were synthesized, and Myc expression levels were evaluated by using quantitative real-time PCR. Myc mRNA levels were normalized to β-actin gene levels ( n = 3 mice analyzed per genotype, with 12 total PCR replicates per sample). Errors are standard errors of the means (***, P
    Epidermal Growth Factor Egf, supplied by PeproTech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/epidermal growth factor egf/product/PeproTech
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    epidermal growth factor egf - by Bioz Stars, 2021-03
    96/100 stars
      Buy from Supplier

    N/A
    EGF L7 Epidermal Growth Factor Like Protein 7 Multiple EGF Like Domains Protein 7 VE Statin is a multi domain protein containing two EGF like domains and one EMI domain
      Buy from Supplier

    Image Search Results


    Average cumulative migration of keratinocytes on gradients of immobilized SS-EGF, SS-IGF-1, and SS-IGF-1+SS-EGF and TCPS over 7 days. (a) g2 (power law) gradient patterns of SS-EGF (150 μg/ml), SS-IGF-1 patterned on SS-EGF (each 75 μg/ml

    Journal:

    Article Title: Co-immobilization of gradient-patterned growth factors for directed cell migration

    doi: 10.1007/s10439-008-9581-1

    Figure Lengend Snippet: Average cumulative migration of keratinocytes on gradients of immobilized SS-EGF, SS-IGF-1, and SS-IGF-1+SS-EGF and TCPS over 7 days. (a) g2 (power law) gradient patterns of SS-EGF (150 μg/ml), SS-IGF-1 patterned on SS-EGF (each 75 μg/ml

    Article Snippet: Recombinant human EGF and IGF-1(Peprotech, Inc., Rocky Hill, NJ) were separately rendered photoactive via conjugation to Sulfo-SANPAH (sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino]hexanoate; Pierce Biotechnology, Inc., Rockford, IL).

    Techniques: Migration

    Mitogen expansion of neurospheres derived from human elderly autopsies of the SVZ. ( A ) Microglia-depleted, SVZ single-cell suspensions treated with hEGF (20 ng/ml) and hFGF-b (10 ng/ml) in serum-free medium (see Materials and Methods) adhere initially

    Journal:

    Article Title: Subventricular Zone Neural Progenitors from Rapid Brain Autopsies of Elderly Subjects with and without Neurodegenerative Disease

    doi: 10.1002/cne.22040

    Figure Lengend Snippet: Mitogen expansion of neurospheres derived from human elderly autopsies of the SVZ. ( A ) Microglia-depleted, SVZ single-cell suspensions treated with hEGF (20 ng/ml) and hFGF-b (10 ng/ml) in serum-free medium (see Materials and Methods) adhere initially

    Article Snippet: Briefly, 10 ml of the single-cell suspensions being passed to secondary flasks (i.e., depleted of microglia) was gently pelleted, washed with proprietary, serum-free neurosphere proliferation medium (SFNPM; NeuroCult® NS-A Proliferation Kit; StemCell Technologies, Vancouver, Canada), and resuspended in T-25 flasks (Corning) in SFNPM supplemented with 20 ng/ml recombinant human epidermal growth factor (hEGF), 10 ng/ml human fibroblast growth factor-basic (hFGF-b) (both from Peprotech, Rocky Hill, NJ), and 2 μg/ml heparin (StemCell Technologies).

    Techniques: Derivative Assay

    The Myc 3′ WRE is required for mitogen-induced Myc gene expression. The proliferative regions of colonic crypts were isolated from 7-week-old Myc 3′ WRE −/− mice and WT littermates and were not treated (Ctrl.) or treated for 1 or 3 h with recombinant Wnt3A, R-spondin1, and EGF (A), Wnt3A and R-spondin1 (B), or EGF (C). RNAs were isolated, cDNAs were synthesized, and Myc expression levels were evaluated by using quantitative real-time PCR. Myc mRNA levels were normalized to β-actin gene levels ( n = 3 mice analyzed per genotype, with 12 total PCR replicates per sample). Errors are standard errors of the means (***, P

    Journal: Molecular and Cellular Biology

    Article Title: The Myc 3′ Wnt-Responsive Element Suppresses Colonic Tumorigenesis

    doi: 10.1128/MCB.00969-13

    Figure Lengend Snippet: The Myc 3′ WRE is required for mitogen-induced Myc gene expression. The proliferative regions of colonic crypts were isolated from 7-week-old Myc 3′ WRE −/− mice and WT littermates and were not treated (Ctrl.) or treated for 1 or 3 h with recombinant Wnt3A, R-spondin1, and EGF (A), Wnt3A and R-spondin1 (B), or EGF (C). RNAs were isolated, cDNAs were synthesized, and Myc expression levels were evaluated by using quantitative real-time PCR. Myc mRNA levels were normalized to β-actin gene levels ( n = 3 mice analyzed per genotype, with 12 total PCR replicates per sample). Errors are standard errors of the means (***, P

    Article Snippet: For experiments involving mitogen treatments, the proliferative compartments of the crypts were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and incubated on a rocking platform in the presence or absence of 100 ng/ml Wnt3A (catalog number 315-20; Peprotech), 500 ng/ml R-spondin1 (catalog number 3474-RS; R & D), and 20 ng/ml epidermal growth factor (EGF) (catalog number 315-09; Peprotech) for 1 or 3 h at 37°C.

    Techniques: Expressing, Isolation, Mouse Assay, Recombinant, Synthesized, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Mitogens induce an exchange of transcriptional regulatory complexes at Myc 5′ and 3′ Wnt-responsive elements. (A) Diagram of the Myc locus, with the Myc 5′ and 3′ WREs indicated by white rectangles. Gray rectangles indicate the positions of the amplicons produced in quantitative real-time PCRs in panels B to G. (B) The proliferative region of colonic crypts were isolated from 7-week-old WT mice and were untreated (Ctrl.) or treated with recombinant Wnt3A, EGF, and R-spondin1 (mitogens) for 3 h. ChIP assays were performed by using antibodies directed against RNA polymerase 2 (RNAP), and the precipitated DNA was measured by using quantitative real-time PCR with oligonucleotides that hybridized to the regions indicated. Colonic crypts were isolated from 3 mice, and 12 PCR replicates were prepared for each sample. Errors are standard errors of the means (**, P

    Journal: Molecular and Cellular Biology

    Article Title: The Myc 3′ Wnt-Responsive Element Suppresses Colonic Tumorigenesis

    doi: 10.1128/MCB.00969-13

    Figure Lengend Snippet: Mitogens induce an exchange of transcriptional regulatory complexes at Myc 5′ and 3′ Wnt-responsive elements. (A) Diagram of the Myc locus, with the Myc 5′ and 3′ WREs indicated by white rectangles. Gray rectangles indicate the positions of the amplicons produced in quantitative real-time PCRs in panels B to G. (B) The proliferative region of colonic crypts were isolated from 7-week-old WT mice and were untreated (Ctrl.) or treated with recombinant Wnt3A, EGF, and R-spondin1 (mitogens) for 3 h. ChIP assays were performed by using antibodies directed against RNA polymerase 2 (RNAP), and the precipitated DNA was measured by using quantitative real-time PCR with oligonucleotides that hybridized to the regions indicated. Colonic crypts were isolated from 3 mice, and 12 PCR replicates were prepared for each sample. Errors are standard errors of the means (**, P

    Article Snippet: For experiments involving mitogen treatments, the proliferative compartments of the crypts were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and incubated on a rocking platform in the presence or absence of 100 ng/ml Wnt3A (catalog number 315-20; Peprotech), 500 ng/ml R-spondin1 (catalog number 3474-RS; R & D), and 20 ng/ml epidermal growth factor (EGF) (catalog number 315-09; Peprotech) for 1 or 3 h at 37°C.

    Techniques: Produced, Isolation, Mouse Assay, Recombinant, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Long chain n-3 PUFA attenuate Ras mediated ERK signaling (A) SW48-KRasG12D or (C) DKOB8 cells were grown in 8-well glass bottom dishes and treated with 50 μM indicated fatty acid for 72 h. (A) SW48-KRasG12D cells were serum starved (0% FBS) over the final 18 h in the presence of fatty acid and stimulated with EGF (25 ng/ml) for 5 min, then immediately fixed in ice cold 100% methanol. (C) DKOB8 cells were incubated with ponasterone A (10 μM) for the final 48 h, and were serum starved (0% FBS) over the final 18 h in the presence of fatty acid and ponasterone A, then immediately fixed in ice cold 100% methanol. Masked images of pERK (A) +/− EGF or (C) +/− ponasterone A. Scale bar = 20 μm. (B, D) Average fluorescence intensity for pERK. Data represent average fluorescence intensity from at least 10 fields of view with ~10-30 cells per field, from 3 independent experiments. (E) Representative immunofluorescence confocal images of DKOB8 cells stained for pERK (red) and nuclei (blue). (F) Quantification of the ratio of fluorescence intensity of pERK in the nucleus vs cytoplasm. Data represent average fluorescence intensity ratio of nucleus to cytoplasm for at least 90 cells, from 3 independent experiments. Statistical significance between treatments as indicated by different letters (P

    Journal: Cancer research

    Article Title: Long chain n-3 fatty acids attenuate oncogenic KRas-driven proliferation by altering plasma membrane nanoscale proteolipid composition

    doi: 10.1158/0008-5472.CAN-18-0324

    Figure Lengend Snippet: Long chain n-3 PUFA attenuate Ras mediated ERK signaling (A) SW48-KRasG12D or (C) DKOB8 cells were grown in 8-well glass bottom dishes and treated with 50 μM indicated fatty acid for 72 h. (A) SW48-KRasG12D cells were serum starved (0% FBS) over the final 18 h in the presence of fatty acid and stimulated with EGF (25 ng/ml) for 5 min, then immediately fixed in ice cold 100% methanol. (C) DKOB8 cells were incubated with ponasterone A (10 μM) for the final 48 h, and were serum starved (0% FBS) over the final 18 h in the presence of fatty acid and ponasterone A, then immediately fixed in ice cold 100% methanol. Masked images of pERK (A) +/− EGF or (C) +/− ponasterone A. Scale bar = 20 μm. (B, D) Average fluorescence intensity for pERK. Data represent average fluorescence intensity from at least 10 fields of view with ~10-30 cells per field, from 3 independent experiments. (E) Representative immunofluorescence confocal images of DKOB8 cells stained for pERK (red) and nuclei (blue). (F) Quantification of the ratio of fluorescence intensity of pERK in the nucleus vs cytoplasm. Data represent average fluorescence intensity ratio of nucleus to cytoplasm for at least 90 cells, from 3 independent experiments. Statistical significance between treatments as indicated by different letters (P

    Article Snippet: For quantitative phospho-ERK analysis, cells were seeded in cell imaging 8 chamber coverglass slides (Eppendorf, 0030742036) and treated with select fatty acids (50 μM) for 72 h. Cells were subsequently serum starved (0% FBS) for 18 h in the presence of fatty acid, stimulated with EGF (25 ng/ml) (PeproTech, 315-09) for 5 min and immediately fixed in ice cold 100% methanol.

    Techniques: Incubation, Fluorescence, Immunofluorescence, Staining