Journal: Oncotarget

Article Title: Emx2 as a novel tool to suppress glioblastoma

doi: 10.18632/oncotarget.9322

Figure Lengend Snippet: Persisting antioncogenic efficacy of Emx2 upon neural nestin enhancer-restricted overexpression In vitro kinetic progression of GbmA and GbmB lines C, D. , engineered by lentiviral vectors and TetON technology as in A, B. , and kept as floating cultures, under Fgf2 and Egf. Cell numbers were normalized against t =0 values. n is the number of biological replicates. p -value was calculated by t-test (one-tail, unpaired): *** p

Article Snippet: In both cases, mediums were supplemented with 1X Penicillin/Streptomycin, 2 μg/ml human heparin (StemCell Technologies #07980), 20 ng/ml recombinant human EGF (ThermoFisher #PHG0311), 20 ng/ml recombinant human FGF2 (ThermoFisher #PHG0261).

Techniques: Over Expression, In Vitro

Journal: Oncotarget

Article Title: Emx2 as a novel tool to suppress glioblastoma

doi: 10.18632/oncotarget.9322

Figure Lengend Snippet: Population dynamics of Emx2 gain-of-function GBM cultures In vitro kinetic progression of U87MG, T98G, GbmA, GbmB, GbmC, GbmD and GbmE GBM lines C-I ., engineered by lentiviral vectors and TetON technology as in A, B. , and kept as adherent C, D. or floating cultures E-I. , under Fgf2 and Egf. Ki67 + proliferating L, M. and activated-Casp3 + apoptotic N, O. fractions of GbmA, GbmB and GbmC glioblastoma cells, engineered by control ( J. , a-b1) and Emx2 -GOF (J, a-b2) lentiviral sets, and kept as floating cultures according to the timetable in K. Cell numbers were normalized against t =0 values (C-I), or control values L, N. [As for (L, N), absolute average control cell frequencies were: 0.207, 0.155 and 0.131 (Ki67 + , in GbmA, GbmB and GbmC cultures, respectively); 0.001, 0.012 and 0.012 (actCasp3 + , in GbmA, GbmB and GbmC cultures, respectively)]. n is the number of biological replicates. p -value was calculated by t-test (one-tail, unpaired): * p

Article Snippet: In both cases, mediums were supplemented with 1X Penicillin/Streptomycin, 2 μg/ml human heparin (StemCell Technologies #07980), 20 ng/ml recombinant human EGF (ThermoFisher #PHG0311), 20 ng/ml recombinant human FGF2 (ThermoFisher #PHG0261).

Techniques: In Vitro

Journal: Oncotarget

Article Title: USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

doi:

Figure Lengend Snippet: (a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 3.2 nM EGF Alexa Fluor 555. After 15 min the cells were acid washed where indicated, fixed and the nuclei stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was assessed in brightfield and fluorescent images taken using confocal microscopy. The right hand panels are enlarged images of the indicated area in the left hand panels. Scale bar = 10 μm. (b) HeLa cells were transfected with either scrambled shRNA or USP17 shRNA1 and 72 hrs post transfection the cells were starved in serum free medium for 3hrs prior to incubation with either 0 nM, 0.32 nM or 3.2 nM recombinant EGF. After 15mins the cells were washed, fixed and stained using an anti-EGFR antibody. EGFR (green) internalisation was assessed in fluorescent images taken using confocal microscopy. The right hand panels are enlarged images of the indicated area in the left hand panels. White arrows point out plasma membrane staining for EGFR. Scale bar = 20 μm.

Article Snippet: EGF internalisation Transfected cells were rested for 3hrs in DMEM medium without serum and stimulated with 0.32nM-3.2nM EGF Alexa Fluor 555 (Invitrogen-Molecular Probes, Eugene, USA) or recombinant EGF (Invitrogen-Gibco, Maryland, USA) for the indicated times in the figures.

Techniques: Transfection, Incubation, Staining, Confocal Microscopy, shRNA, Recombinant

Journal: Oncotarget

Article Title: USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

doi:

Figure Lengend Snippet: (a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 where indicated. After 15 min the cells were acid washed, where indicated, fixed and the nuclei stained with DAPI. The cells were then stained using an anti-EGFR antibody and EGF Alexa Fluor 555 (red) and EGFR (green) internalisation was assessed in brightfield and fluorescent images taken using confocal microscopy. The right panels are enlarged images of the indicated area in the left panels. Scale bar = 10 μm. (b) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 and EGFR co-staining post acid wash. (c) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF. Whole cell lysates were harvested and levels of phosphorylated ERK1/2, ERK1/2, EGFR and tubulin were assessed by immuno-blotting using anti-pERK1/2, anti-ERK1/2, anti-EGFR and anti-tubulin antibodies. ** p

Article Snippet: EGF internalisation Transfected cells were rested for 3hrs in DMEM medium without serum and stimulated with 0.32nM-3.2nM EGF Alexa Fluor 555 (Invitrogen-Molecular Probes, Eugene, USA) or recombinant EGF (Invitrogen-Gibco, Maryland, USA) for the indicated times in the figures.

Techniques: Transfection, Incubation, Staining, Confocal Microscopy

Journal: Oncotarget

Article Title: USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

doi:

Figure Lengend Snippet: (a) HeLa cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 and mRNA and protein samples were harvested at the time points indicated. USP17 and GAPDH (loading control) mRNA were then assessed by RT-PCR. USP17 protein levels were assessed by immunoblotting and tubulin was used as a loading control. (b) HeLa cells were transfected as indicated. 72 hrs post transfection mRNA and protein samples were harvested and USP17 mRNA and protein levels assessed as above. (c) HeLa cells were transfected with the constructs indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555. After 15 min the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The bottom panels are enlarged images of the indicated area in the top panels. Scale bar = 20 μm. (d) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 internalisation. (e) A549 cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3hrs prior to incubation with 0.32nM EGF Alexa Fluor 555. After 10 mins the cells were either acid washed or washed with PBS. Subsequently they were fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The middle panels are enlarged images of the indicated area in the upper panels. Scale bar = 25 μm. (f) HeLa cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32nM EGF Alexa Fluor 555. After 45 mins the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The lower panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm. ** p

Article Snippet: EGF internalisation Transfected cells were rested for 3hrs in DMEM medium without serum and stimulated with 0.32nM-3.2nM EGF Alexa Fluor 555 (Invitrogen-Molecular Probes, Eugene, USA) or recombinant EGF (Invitrogen-Gibco, Maryland, USA) for the indicated times in the figures.

Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Staining, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: Histone deacetylase 1 and 2 differentially regulate apoptosis by opposing effects on extracellular signal-regulated kinase 1/2

doi: 10.1038/cddis.2010.21

Figure Lengend Snippet: HDAC2 is important for maintaining ERK1/2 activation levels to keep cell survival. The basal levels of ERK1/2 activation were examined by immunoblotting in iHDAC1-S1, iHDAC2 and empty vector-transfected cells ( a ). The iHDAC2 cells were cultured in the presence or absence of 100 ng/ml EGF. The p-ERK1/2 levels were examined by immunoblotting 10 min after the treatment ( b ), and apoptosis were determined by FACS 72 h after the treatment ( c ). The iHDAC2 cells were treated with 20 ng/ml TSA ( d , e ) or 5 μ M MS-275 ( f , g ). The p-ERK1/2 levels were examined by immunoblotting 8 h after the treatment, apoptosis were determined by FACS 72 h after the treatment. The data represent the means±S.D. of at least three independent experiments

Article Snippet: TSA, NaBu, MS-275, Ac-DEVD-pNA (caspase-3 substrate) and human recombinant EGF were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Activation Assay, Plasmid Preparation, Transfection, Cell Culture, FACS, Mass Spectrometry

Journal: Molecular cancer research : MCR

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

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

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

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

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

Journal: Molecular cancer research : MCR

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

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

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

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

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

Journal:

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

doi: 10.1167/iovs.06-0560

Figure Lengend 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

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).

Techniques:

Journal:

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

doi: 10.1167/iovs.06-0560

Figure Lengend 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

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).

Techniques: Cell Culture

Journal:

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

doi: 10.1167/iovs.06-0560

Figure Lengend 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)

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).

Techniques:

Journal:

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

doi: 10.1167/iovs.06-0560

Figure Lengend 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

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).

Techniques:

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

Journal: EBioMedicine

Article Title: Prokineticin 2 Plays a Pivotal Role in Psoriasis

doi: 10.1016/j.ebiom.2016.10.022

Figure Lengend Snippet: (a b) PK2 accelerated inflammatory phase during wound healing. a. Histopathological examination of mice wound treated by PK2/EGF. (25 ×: visual field of magnified 25 times ; 100 ×: visual field of magnified 100 times). b. The statistic of inflammatory cells in wound.

Article Snippet: Ten micro liters of vehicle (0.9% NaCl solution), PK2 (100 μg/ml) or recombinant murine EGF (100 μg/ml, PeproTech Inc., USA, positive control) was applied directly to the wound site twice daily for seven days (day 1–day 7).

Techniques: Mouse Assay

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

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

Journal: International Journal of Molecular Sciences

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

doi: 10.3390/ijms18010148

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

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

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

Journal: eLife

Article Title: Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes

doi: 10.7554/eLife.06156

Figure Lengend Snippet: Redistribution of endosomal EGF is sufficient to increase MAPK activation in PC12 cells. ( A – D ) Analysis of MAPK activation in PC12 cells after partial protein depletion of either EEA1, Rabenosyn5, and Vps45 or EEA1, Syntaxin-6, and Syntaxin-13, or mock treatment and stimulation with 100 ng/ml EGF or 50 ng/ml NGF for 30 min ( A ) Representative images of Erk1/2 activation by immunofluorescence in PC12 cells. phospho-Erk1/2 is shown in green and nuclei are shown in blue. Scale bars, 10 μm. ( B ) Increase in phospho-Erk1/2 intensity compared to EGF-treated control cells. The total intensity was normalized by the fraction of the area covered by cells. ( C ) Representative images of c-Fos phosphorylation by immunofluorescence in PC12 cells. phospho-c-Fos is shown in green. Scale bar, 25 μm. ( D ) Increase in nuclear phospho-c-Fos intensity compared to EGF-treated control cells. In all cases, data show mean ± SEM. For each parameter, pair-wise comparisons were done against EGF-stimulated mock-treated cells. *p

Article Snippet: PC12 cells were starved for 36 hr before stimulation either with 100 ng/ml EGF (Invitrogen) or 50 ng/ml NGF (R & D Systems) for 30 min. E14.5 Dlk1+ hepatoblasts were starved for 24 hr before stimulation with either 10 ng/ml EGF or HGF (R & D systems).

Techniques: Activation Assay, Immunofluorescence

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0165419

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

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

Techniques:

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0165419

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

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

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: E5 Protein of Human Papillomavirus Type 16 Protects Human Foreskin Keratinocytes from UV B-Irradiation-Induced Apoptosis

doi: 10.1128/JVI.76.1.220-231.2002

Figure Lengend Snippet: HPV16 E5 enhances the activation of Akt and ERK1/2 in HFKs after UV B irradiation in the presence of EGF. LXSN-infected and L(16E5)SN-infected cells were grown in K-SFM without EGF for 24 h, either stimulated or not with 5 ng of EGF per ml for 1 min, and then irradiated with 0 and 400 J of UV B per m 2 . In parallel, some cells were pretreated with 1 μM wortmannin (WO) or 50 μM PD98059 (PD) for 1 h. All cells were lysed 15 min after UV B irradiation. Cell lysates containing 40 μg of protein were separated on an SDS-12% polyacrylamide gel, transferred to nitrocellulose, and analyzed with antibodies to phospho-Akt (p-Akt) and total Akt (A) and phospho-ERK1/2 and total ERK1/2 (B).

Article Snippet: The frozen cells were subsequently thawed in complete serum-free medium (K-SFM; GIBCO/BRL) supplemented with human recombinant EGF (GIBCO/BRL) and bovine pituitary extract (GIBCO/BRL).

Techniques: Activation Assay, Irradiation, Infection

Journal: American Journal of Physiology - Cell Physiology

Article Title: Epidermal growth factor inhibits intestinal NHE8 expression via reducing its basal transcription

doi: 10.1152/ajpcell.00081.2010

Figure Lengend Snippet: Signaling pathway studies of EGF regulation of hNHE8 gene expression. Caco-2 cells were transfected with promoter construct pGL3/-32 bp and pretreated with various inhibitors for 2 h before EGF was added. Relative change is shown as the ratio of luciferase activity in EGF-treated cells to luciferase activity in vehicle-treated cells in the presence or absence of various inhibitors. Results are means ± SE from 4 independent experiments. * P

Article Snippet: To test the effect of EGF on hNHE8 promoter activity, transfected cells were treated with 100 ng/ml human recombinant EGF for 18 h before promoter reporter assay.

Techniques: Expressing, Transfection, Construct, Luciferase, Activity Assay

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

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

doi: 10.1073/pnas.141222298

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

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

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

Journal: The Journal of Biological Chemistry

Article Title: Ectodysplasin A protein promotes corneal epithelial cell proliferation

doi: 10.1074/jbc.M117.803809

Figure Lengend Snippet: Eda activated EGF-EGFR signaling pathway in HCE cells. A, HCE cells were cultured with different concentrations of Eda (0, 5, 10, and 20 ng/ml). CCK8 assay results showed 5 and 10 ng/ml of Eda-enhanced cell proliferation, and reached a plateau at 10 ng/ml ( n = 4, **, p

Article Snippet: To delineate the contribution made by Eda to the wound healing response, 8-week-old wild-type mice eyeballs that had a 2.0-mm corneal epithelial debridement wound on the central cornea were cultured in DMEM + 2% FBS in the absence or presence of 20 ng/ml of mouse recombinant Eda protein or 10 ng/ml of mouse recombinant EGF protein (PMG8041, Life Technologies) for different durations from 12 to 36 h. Sodium fluorescein staining evaluated the wound closure process during these periods ( A ).

Techniques: Cell Culture, CCK-8 Assay

Journal: The Journal of Biological Chemistry

Article Title: Ectodysplasin A protein promotes corneal epithelial cell proliferation

doi: 10.1074/jbc.M117.803809

Figure Lengend Snippet: Eda promotes corneal epithelial wound healing during in vitro organ culture. A, 8-week-old wild-type mice eyeballs with a 2.0-mm corneal epithelial debridement wound on the central cornea were cultured in DMEM + 2% FBS in the absence or presence of 20 ng/ml of recombinant Eda protein or 10 ng/ml of EGF protein for different durations from 12 to 36 h. Fluorescein staining evaluated differences in wound closure extent during the culture. B, the wound closure extent reached 26.5% in the Eda treatment group ( Eda ), 28.7% in the EGF treatment group ( EGF ), whereas it was 9.6% in the control group (NC) after 12 h culture. At 24 h, the wounds had healed to 80.0% in the Eda treatment group, whereas it was 58.7% in the control group, and completed healed in the EGF group. At 36 h, the epithelial defect was completely healed in both groups ( n = 4, *, p

Article Snippet: To delineate the contribution made by Eda to the wound healing response, 8-week-old wild-type mice eyeballs that had a 2.0-mm corneal epithelial debridement wound on the central cornea were cultured in DMEM + 2% FBS in the absence or presence of 20 ng/ml of mouse recombinant Eda protein or 10 ng/ml of mouse recombinant EGF protein (PMG8041, Life Technologies) for different durations from 12 to 36 h. Sodium fluorescein staining evaluated the wound closure process during these periods ( A ).

Techniques: In Vitro, Organ Culture, Mouse Assay, Cell Culture, Recombinant, Staining

Journal: Molecular and Cellular Biology

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

doi: 10.1128/MCB.25.17.7734-7742.2005

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

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

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

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

Article Title: Maternal activation of the EGFR prevents translocation of gut-residing pathogenic Escherichia coli in a model of late-onset neonatal sepsis

doi: 10.1073/pnas.1912022117

Figure Lengend Snippet: Inhibition of EGFR results in bacterial translocation of gut-residing E. coli . ( A ) Concentration of EGF as measured by ELISA from the stool of children fed mother’s own milk (green squares) or formula (gray circles). In B – F , conventionally reared mice were gavaged with 2 × 10 5 CFUs of E. coli and injected with EGFRi on DOL5. ( B ) CFUs in stool following gavage of 2 × 10 5 CFUs of E. coli C1 nalR (blue) or BSI-A nalR (red) in conventionally reared mice on DOL5. ( C ) CFUs in mesenteric lymph node (MLN), spleen, and liver 3 d following gavage of 2 × 10 5 CFUs of E. coli C1 nalR (blue) or BSI-A nalR (red) on DOL5 in conventionally reared mice injected with tyrphostin AG1478 (EGFRi) or vehicle. ( D ) Neutrophils in the blood 48 h following gavage of 2 × 10 5 CFUs of E. coli C1 nalR or E. coli BSI-A nalR and injected with EGFRi as indicated or vehicle. ( E ) Survival of mice following vehicle or EGFRi injection and gavage of 2 × 10 5 CFUs of E. coli C1 nalR or BSI-A nalR . ( F ) Weight of mice following gavage of 2 × 10 5 CFUs of E. coli BSI-A nalR and injected with EGFRi (black), or vehicle (green). Red dots denote moribund pups. n = 5 individuals in each group with multiple time points in A . n = 5 mice per group in B – D . n = 15 mice per group from 3 independent litters in E . n = 10 mice per group in F . Individual data points plotted in B – D with mean and SD plotted per group. Statistics used: Linear regression ( A ), Mann–Whitney ( C ), one-way ANOVA ( D ), two-way ANOVA ( F ), Kaplan–Meier ( E ), *denotes statistical significance, P

Article Snippet: Frozen stool specimens were resuspended in PBS, homogenized, and analyzed by enzyme-linked immunosorbent assay (ELISA) for human EGF (R & D Systems), human amphiregulin (AREG) (R & D Systems), human TGF-α (R & D Systems), and human heparin-binding EGF (HB-EGF) (R & D Systems), per the manufacturer’s protocol.

Techniques: Inhibition, Translocation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, MANN-WHITNEY

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

Article Title: Maternal activation of the EGFR prevents translocation of gut-residing pathogenic Escherichia coli in a model of late-onset neonatal sepsis

doi: 10.1073/pnas.1912022117

Figure Lengend Snippet: Luminal EGF protects against bacterial translocation and susceptibility to bloodstream infections. ( A ) CFUs in lamina propria of small intestine or colon following gavage of 2 × 10 5 CFUs of E. coli BSI-A nalR in ACF mice. ( B ) Counts of GAPs per colon crypt in SCF or ACF mice and gavaged with EGF (10 ng/mL) or vehicle. ( C ) Concentration of phosphorylated EGFR per colon epithelial fraction as measured by ELISA. GAPs (black), ACF given a single gavage of recombinant murine EGF at the time of infection (red), or ACF mice given daily gavages of EGF (green). ( D ) Percentage of GCs or IECs containing E. coli BSI-A nalR . ( E ) CFUs in stool, mesenteric lymph node (MLN), spleen, and liver in the days following gavage of 2 × 10 5 CFUs of E. coli BSI-A nalR in ACF mice treated with EGF. ( F ) Survival of ACF mice following gavage of 2 × 10 5 CFUs of E. coli BSI-A nalR in ACF mice (black), ACF gavaged with EGF (10 ng/mL) at the time of infection (red), or ACF mice given daily gavages of EGF (10 ng/mL) (green). n = 4 mice in A , n = 4 mice in B – D . n = 5 in E . n = 6 mice per group in F . Individual data points plotted in A – E with mean and SD plotted per group. Statistics used: one-way ANOVA ( A – E ), Kaplan–Meier ( F ), *denotes statistical significance, P

Article Snippet: Frozen stool specimens were resuspended in PBS, homogenized, and analyzed by enzyme-linked immunosorbent assay (ELISA) for human EGF (R & D Systems), human amphiregulin (AREG) (R & D Systems), human TGF-α (R & D Systems), and human heparin-binding EGF (HB-EGF) (R & D Systems), per the manufacturer’s protocol.

Techniques: Translocation Assay, Mouse Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Infection

Journal: Scientific Reports

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

doi: 10.1038/srep43771

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

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

Techniques: CCK-8 Assay, Cell Culture, Recombinant

Journal: Nucleic Acids Research

Article Title: Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

doi: 10.1093/nar/gky145

Figure Lengend Snippet: Overexpression of EGFR increases PS-ASO activity in HEK cells. ( A ) Western analyses for proteins in control or EGFR overexpressing HEK cells treated with EGF for indicated time. The blot was probed sequentially with different antibodies detecting phosphorylated EGFR (P-EGFR), total EGFR (T-EGFR) and total ERK (T-ERK) as loading control. ( B ) Representative images of immunofluorescent staining for EGFR (green) in HEK cells overexpressing EGFR. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 5 μm. ( C ) Reduction of Malat1 RNA was quantified by qRT-PCR in HEK cells with or without overexpression of EGFR. Data are relative to untreated cells. Activity curves were fitted from data plotted in panels based on a non-linear regression model. The error bars represent standard deviations from 3 independent experiments. P

Article Snippet: Purified recombinant EGF (PHG0311L, ThermoFisher Scientific) or EGFR protein (PV3872, ThermoFisher Scientific) were incubated with FITC-labeled 2′-MOE gapmer PS-ASOs (IONIS ID 256903) or phosphodiester ASO (synthesized by Integrated DNA Technologies) in 100 μl binding buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol) as previously described ( ).

Techniques: Over Expression, Allele-specific Oligonucleotide, Activity Assay, Western Blot, Staining, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

doi: 10.1093/nar/gky145

Figure Lengend Snippet: PS-ASOs do not affect EGFR synthesis, degradation and recycling. ( A ) A431 cells were pulse labeled with [ 35 S]-Met/Cys for 50 min. EGFR and S100a10 were immunoprecipitated using their specific antibodies, and analyzed by SDS-PAGE. ( B ) Intracellular levels of nascent EGFR were chased and analyzed by SDS-PAGE at indicated times after the labeling and were visualized and quantified by autoradiography, as in ( A ). S100a10 was detected and used as a loading control. ( C ) Protein samples were analyzed by western analyses from A431 cells treated with EGF or EGF and PS-ASOs. The blot was probed sequentially with antibodies specific to the various proteins and the images were quantified by ImageLab (Bio-Rad). ( D ) Representative images of immunofluorescent staining for EGFR (green) in A431 cells incubated with EGF ( a ) or EGF and Cy3-labeled PS-ASOs (red) ( b-1 and b-2 ) for 16 h, and then 2 h after the removal of EGF ( c ) or EGF and Cy3-labeled PS-ASOs ( d-1 and d-2 ). The nuclei were stained with Hoechst 33342 (blue). Scale bars, 5 μm.

Article Snippet: Purified recombinant EGF (PHG0311L, ThermoFisher Scientific) or EGFR protein (PV3872, ThermoFisher Scientific) were incubated with FITC-labeled 2′-MOE gapmer PS-ASOs (IONIS ID 256903) or phosphodiester ASO (synthesized by Integrated DNA Technologies) in 100 μl binding buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol) as previously described ( ).

Techniques: Labeling, Immunoprecipitation, SDS Page, Autoradiography, Western Blot, Staining, Incubation

Journal: Nucleic Acids Research

Article Title: Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

doi: 10.1093/nar/gky145

Figure Lengend Snippet: PS-ASOs traffic together with EGF and EGFR through the endocytic pathway. ( A ) Representative images of immunofluorescent staining for clathrin and EGFR in A431 cells incubated with non-labeled EGF and Cy3-labeled PS-ASOs for 30 min. The nuclei were stained with Hoechst 33342 (blue). The arrow indicates co-localization between EGFR (green) and PS-ASOs (red); between EGFR (green) and clathrin (red); between PS-ASOs (red) and clathrin (green). Scale bars, 2 μm. ( B ) Representative images of immunofluorescent staining for clathrin in A431 cells incubated with FITC-EGF and Cy3-labeled PS-ASOs for 30 min. The nuclei were stained with Hoechst 33342 (blue). The arrow indicates co-localization between EGF (green) and PS-ASOs (red); between EGF (green) and clathrin (red); between PS-ASOs (red) and clathrin (green). Scale bars, 2 μm. ( C ) Representative images of immunofluorescent staining for EEA1 and EGFR in A431 cells incubated with non-labeled EGF and Cy3-labeled PS-ASOs for 30 min. The nuclei were stained with Hoechst 33342 (blue). The arrow indicates co-localization between EGFR (green) and PS-ASOs (red); between EGFR (green) and EEA1 (red); and between PS-ASOs (red) and EEA1 (green). Scale bars, 2 μm. ( D ) Representative images of immunofluorescent staining for EEA1 in A431 cells incubated with FITC-EGF and Cy3-labeled PS-ASOs for 30 min. The nuclei were stained with Hoechst 33342 (blue). The arrow indicates co-localization between EGF (green) and PS-ASOs (red); between EGF (green) and EEA1 (red); between PS-ASOs (red) and EEA1 (green); Scale bars, 2 μm.

Article Snippet: Purified recombinant EGF (PHG0311L, ThermoFisher Scientific) or EGFR protein (PV3872, ThermoFisher Scientific) were incubated with FITC-labeled 2′-MOE gapmer PS-ASOs (IONIS ID 256903) or phosphodiester ASO (synthesized by Integrated DNA Technologies) in 100 μl binding buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol) as previously described ( ).

Techniques: Staining, Incubation, Labeling

Journal: Oncotarget

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

doi:

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

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

Techniques: Planar Chromatography, Concentration Assay

Journal: Oncotarget

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

doi:

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

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

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

Journal: Sensors (Basel, Switzerland)

Article Title: SirT1--A Sensor for Monitoring Self-Renewal and Aging Process in Retinal Stem Cells

doi: 10.3390/s100606172

Figure Lengend Snippet: Isolation of rat RSCs. (A) In vitro culture of rat RSCs. The floating neurospheres were derived from RSCs and cultivated in serum-free medium with EGF and bFGF. Bars: 100 μm. (B) Comparison of the gene expression among fresh RSCs at baseline, RSC after 7 and 14 day differentiation in vitro culture. The symbol * indicated a significant difference among RSC groups at baseline, day 7 and day 14 differentiation (*p

Article Snippet: Afterwards, the enzyme solution was removed, and viable cells were counted by trypan blue exclusion and plated as 5,000 cells/200 μL per well in 96-well plates (Corning, Acton, MA, USA) replaced with serum-free culture media composed of DMEM/F-12 medium (Gibcol) with insulin (25 μg/mL), transferrin (100 μg/mL), progesterone (20 nM), putrescine (60 μM), sodium selenite (30 nM), and human recombinant EGF 20 ng/mL and bFGF 20 ng/mL (R & D Systems, Minneapolis, MN, USA).

Techniques: Isolation, In Vitro, Derivative Assay, Expressing