Journal:

Article Title: Elevated Levels of Circulating Interleukin-18 in Human Immunodeficiency Virus-Infected Individuals: Role of Peripheral Blood Mononuclear Cells and Implications for AIDS Pathogenesis

doi: 10.1128/JVI.76.24.12448-12456.2002

Figure Lengend Snippet: Effect of TGF-β on IL-18 production from PBMC. PBMC (2 × 105) from HIV-seronegative donors were cultured in 200 μl of the culture medium in 96-well plate alone or in the presence of recombinant human (rh) TGF-β (20 ng per ml; R & D Systems), TGF-β-neutralizing, or control antibody (5 μg per ml each; R & D Systems). Culture supernatants were collected 24 h later and assayed for IL-18 content with the ELISA kit. The figure shows average ± standard error IL-18 contents from three replicate wells. Only the addition of the neutralizing antibody resulted in a significant (P < 0.05) increase in TGF-β production.

Article Snippet: PBMC (2 × 10 5 ) from HIV-seronegative donors were cultured in 200 μl of the culture medium in 96-well plate alone or in the presence of recombinant human (rh) TGF-β (20 ng per ml; R & D Systems), TGF-β-neutralizing, or control antibody (5 μg per ml each; R & D Systems).

Techniques: Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Elevated Levels of Circulating Interleukin-18 in Human Immunodeficiency Virus-Infected Individuals: Role of Peripheral Blood Mononuclear Cells and Implications for AIDS Pathogenesis

doi: 10.1128/JVI.76.24.12448-12456.2002

Figure Lengend Snippet: TGF-β contents of the plasma samples. The level of TGF-β was measured in the samples with a commercial ELISA kit. The figure depicts average ± standard error concentration of this cytokine in the samples after its activation (total TGF-β). N and P, HIV-seronegative healthy and HIV-infected donors, respectively. The star on the top of a bar indicates a significant (P < 0.05) difference between the two group means.

Article Snippet: PBMC (2 × 10 5 ) from HIV-seronegative donors were cultured in 200 μl of the culture medium in 96-well plate alone or in the presence of recombinant human (rh) TGF-β (20 ng per ml; R & D Systems), TGF-β-neutralizing, or control antibody (5 μg per ml each; R & D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Activation Assay, Infection

Journal:

Article Title: Elevated Levels of Circulating Interleukin-18 in Human Immunodeficiency Virus-Infected Individuals: Role of Peripheral Blood Mononuclear Cells and Implications for AIDS Pathogenesis

doi: 10.1128/JVI.76.24.12448-12456.2002

Figure Lengend Snippet: Correlation between the levels of TGF-β in plasma and IL-18 in serum of HIV-infected individuals. The figure depicts a significant negative correlation between these two parameters as determined by the Pearson's method.

Article Snippet: PBMC (2 × 10 5 ) from HIV-seronegative donors were cultured in 200 μl of the culture medium in 96-well plate alone or in the presence of recombinant human (rh) TGF-β (20 ng per ml; R & D Systems), TGF-β-neutralizing, or control antibody (5 μg per ml each; R & D Systems).

Techniques: Infection

Journal: PLoS ONE

Article Title: Py2T Murine Breast Cancer Cells, a Versatile Model of TGFβ-Induced EMT In Vitro and In Vivo

doi: 10.1371/journal.pone.0048651

Figure Lengend Snippet: ( A ) Primary tumor cells were isolated from an advanced breast tumor of a MMTV-PyMT transgenic female mouse and were cultured for at least 2 months prior to further experimentation, resulting in a novel cell line termed Py2T. ( B ) Py2T cells maintain the MMTV-PyMT transgene. The MMTV-PyMT transgene was detected by PCR and agarose gel electrophoresis. DNA from an MMTV-PyMT tumor and from normal murine mammary gland (NMuMG) cells served as positive and negative controls, respectively. ( C ) Py2T cells lost the expression of the MMTV-PyMT transgene. Immunoblotting for the PyMT protein was performed on lysates of Py2T cells untreated or treated with 0.1 µM Dexamethasone for up to 72 h to induce the MMTV promoter. Lysates of an MMTV-PyMT tumor and NMuMG cells served as positive and negative controls, respectively. ( D ) Treatment of Py2T cells with known EMT inducers. Cells were continuously treated with the indicated growth factors and cytokines for 10 days (2 ng/mL TGFβ1; 50 ng/mL EGF; 10 ng/mL IGF-I; 50 ng/mL HGF; 20 ng/mL FGF-2; 20 ng/mL PDGF-BB; 50 ng/mL IL-6). Potential morphological changes were analyzed by phase-contrast microscopy. ( E ) Expression of epithelial (E-cadherin) and mesenchymal (N-cadherin, fibronectin) markers were analyzed by immunoblotting of the lysates of cells treated in (D). ( F ) Immunoblotting analysis of EMT marker expression in Py2T and Py2T LT cells. The mesenchymal subline Py2T LT (long-term) was generated by TGFβ-treatment of Py2T cells for at least 20 days, and was subsequently maintained in TGFβ containing growth medium. ( G ) Analysis of markers for EMT and breast cell type before and after TGFβ-induced EMT. Immunofluorescence staining was performed with antibodies against E-Cadherin (epithelial marker), vimentin (mesenchymal marker), estrogen receptor alpha (ERα), cytokeratin 8/18 (luminal markers) and cytokeratin 14 (basal marker). Scale bar, 20 µm.

Article Snippet: Reagents: recombinant human (rh) TGFβ1 (240-B-010, R&D Systems), recombinant mouse (rm) EGF (PMG8041, Invitrogen), rmIGF-1 (250-19, Peprotech), rmHGF (2207-HG, R&D Systems), rmbasicFGF (3139-FB-025, R&D Systems), rhPDGF-BB (220-BB, R&D Systems, rhIL-6 (200-06 Peprotech), Dexamethasone (800-437-7500, Chemicon), Matrigel, growth factor reduced (356230, BD), SB431542 (S4317, Sigma Aldrich), SB203580 (ALX-270-339, Axxora), SP600125 (ALX-270-339, Axxora), PD98059 (ALX-385-023, Axxora).

Techniques: Isolation, Transgenic Assay, Cell Culture, Agarose Gel Electrophoresis, Expressing, Western Blot, Microscopy, Marker, Generated, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Py2T Murine Breast Cancer Cells, a Versatile Model of TGFβ-Induced EMT In Vitro and In Vivo

doi: 10.1371/journal.pone.0048651

Figure Lengend Snippet: ( A ) Morphological changes of Py2T cells during a time-course of TGFβ-treatment. Cells were cultured in growth medium containing TGFβ (2 ng/ml) and phase-contrast microscopy pictures were taken at the indicated times. ( B ) Immunoblotting analysis of lysates prepared from Py2T cells treated as in (A). The expression of epithelial (E-cadherin), mesenchymal (N-cadherin, fibronectin), luminal (CK8/18) and basal (CK14) markers was analyzed. ( C ) Changes in the expression of EMT markers during TGFβ-induced EMT of Py2T cells. Py2T cells were treated for 10 days with TGFβ as described in (A). RNA was extracted at the indicated time points of TGFβ-treatment and quantitative RT-PCR was performed with primers specific for the EMT markers indicated. Expression levels are shown as mean fold difference of untreated cells (0d) ± S.E.M of 5 independent experiments. ( D–E ) Reversibility of TGFβ-induced EMT. Py2T cells were treated with TGFβ for 30 days to induce EMT and were then further cultured without TGFβ for additional 30 days. Phase-contrast microscopy images were taken at the indicated time points (E). E-cadherin expression levels were analyzed throughout the experiment by immunoblotting (F).

Article Snippet: Reagents: recombinant human (rh) TGFβ1 (240-B-010, R&D Systems), recombinant mouse (rm) EGF (PMG8041, Invitrogen), rmIGF-1 (250-19, Peprotech), rmHGF (2207-HG, R&D Systems), rmbasicFGF (3139-FB-025, R&D Systems), rhPDGF-BB (220-BB, R&D Systems, rhIL-6 (200-06 Peprotech), Dexamethasone (800-437-7500, Chemicon), Matrigel, growth factor reduced (356230, BD), SB431542 (S4317, Sigma Aldrich), SB203580 (ALX-270-339, Axxora), SP600125 (ALX-270-339, Axxora), PD98059 (ALX-385-023, Axxora).

Techniques: Cell Culture, Microscopy, Western Blot, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Py2T Murine Breast Cancer Cells, a Versatile Model of TGFβ-Induced EMT In Vitro and In Vivo

doi: 10.1371/journal.pone.0048651

Figure Lengend Snippet: ( A ) Smad-mediated canonical TGFβ signaling is dispensable for early changes in morphology and junction disruption. Cells were transfected with a pool of siRNAs against Smad4 or a non-targeting pool and were then treated or not with TGFβ for 1 day as indicated. Fixed cells were stained for the adherens junction components E-cadherin and β-catenin, or for E-cadherin and the tight junction component ZO-1. Note the relocalization of β-catenin from adherens junctions to the cytoplasm upon TGFβ-treatment. Scale bars, 50 µm. ( B ) Immunoblot analysis of lysates from the experiment described in (A) to control for Smad4 knockdown efficiency. ( C ) Requirement of non-canonical TGFβ signaling pathways on early morphological changes and junction disassembly. Cells were pre-treated for 4 hours with chemical inhibitors of the kinases indicated, and were then treated with TGFβ for 1 day and analyzed as described in (A). Scale bars, 50 µm. ( D ) RhoA expression levels during the early stages of EMT. Cells were treated or not with TGFβ for 1 day and RhoA expression levels were analysed by immunoblotting. ( E ) Importance of RhoA levels for tight- and adherens junction integrity. Epithelial Py2T cells were separately transfected with two different siRNAs targeting RhoA to achieve expression levels comparable to those observed in Py2T cells treated with TGFβ (see D). Cells were stained for the adherens junction components E-cadherin and β-catenin, or for E-cadherin and the tight junction component ZO-1. ( F ) Immunoblotting analysis to determine the RhoA knockdown efficiency in the experiment described in (E).

Article Snippet: Reagents: recombinant human (rh) TGFβ1 (240-B-010, R&D Systems), recombinant mouse (rm) EGF (PMG8041, Invitrogen), rmIGF-1 (250-19, Peprotech), rmHGF (2207-HG, R&D Systems), rmbasicFGF (3139-FB-025, R&D Systems), rhPDGF-BB (220-BB, R&D Systems, rhIL-6 (200-06 Peprotech), Dexamethasone (800-437-7500, Chemicon), Matrigel, growth factor reduced (356230, BD), SB431542 (S4317, Sigma Aldrich), SB203580 (ALX-270-339, Axxora), SP600125 (ALX-270-339, Axxora), PD98059 (ALX-385-023, Axxora).

Techniques: Disruption, Transfection, Staining, Western Blot, Control, Knockdown, Protein-Protein interactions, Expressing

Journal: PLoS ONE

Article Title: Py2T Murine Breast Cancer Cells, a Versatile Model of TGFβ-Induced EMT In Vitro and In Vivo

doi: 10.1371/journal.pone.0048651

Figure Lengend Snippet: ( A ) Boyden chamber migration and invasion assay. Cells were treated with TGFβ for the indicated times (LT = long term treatment, as described in Fig. 1F). 25'000 cells were seeded into migration or invasion chambers in duplicate in the absence or presence of TGFβ and allowed to pass through the membrane pores for 24 hours along an FBS gradient. Invasion chambers were pre-coated with growth-factor reduced Matrigel (BD BioCoat chambers). Cells that passed through the membrane pores were stained with crystal violet and photographed ( bottom panels ) and then counted ( top graphs ). Results are expressed as mean ± S.E.M of three independent experiments. ( B ) Scratch wound healing assay. Cells pre-treated with TGFβ or not as indicated were starved over night and scratch wounds were introduced into confluent monolayers. Scratch wound closure was monitored by an IncuCyte™ live cell imaging system. Black masking represents initial gap width at 0 hours. Note the collective, sheet-like wound closure by untreated Py2T cells in contrast to single cell wound infiltration of TGFβ-treated cells (also see Movies S1 and S2 for live imaging data of this experiment). ( C ) Morphology of epithelial Py2T cells and mesenchymal Py2T LT cells grown on plastic tissue culture dishes (2D) and in Matrigel (4 mg/ml; 3D). Structures were grown for 6 days, and stained directly in Matrigel with antibodies against epithelial E-cadherin and ZO-1 or against mesenchymal vimentin and fibronectin. Immunofluorescence images were acquired by confocal microscopy. Scale bars, 25 µm. ( D ) Three-dimensional reconstruction of confocal imaging stacks from cells grown in Matrigel as described in (A) (See also Movies S5 and S6 for rotating 3D models). Scale bars, 25 µm.

Article Snippet: Reagents: recombinant human (rh) TGFβ1 (240-B-010, R&D Systems), recombinant mouse (rm) EGF (PMG8041, Invitrogen), rmIGF-1 (250-19, Peprotech), rmHGF (2207-HG, R&D Systems), rmbasicFGF (3139-FB-025, R&D Systems), rhPDGF-BB (220-BB, R&D Systems, rhIL-6 (200-06 Peprotech), Dexamethasone (800-437-7500, Chemicon), Matrigel, growth factor reduced (356230, BD), SB431542 (S4317, Sigma Aldrich), SB203580 (ALX-270-339, Axxora), SP600125 (ALX-270-339, Axxora), PD98059 (ALX-385-023, Axxora).

Techniques: Migration, Invasion Assay, Membrane, Staining, Wound Healing Assay, Live Cell Imaging, Imaging, Immunofluorescence, Confocal Microscopy

Journal: Oncotarget

Article Title: Tissue transglutaminase induces Epithelial-Mesenchymal-Transition and the acquisition of stem cell like characteristics in colorectal cancer cells

doi: 10.18632/oncotarget.15370

Figure Lengend Snippet: A . Western blots of whole cell lysates of CRCs showing increased expression of TGFβ1 in whole cell lysates of RKO EV control cells, RKO transduced with TG2 (RKOTG2), SW480 or SW620 transduced with empty vectors (EV) cells or transduced with TG2 shRNA (shRNA). B . TGFβ1 released into cell culture media of CRCs determined by ELISA undertaken as described in the Materials and Methods. Data represent means ± S.D., from two experiments each performed in triplicate (*, **, # and ~, p<0.05). C . Representative Dot blotting of cell culture media of CRCs both EV controls and cell transduced with TG2 (TG2) or shRNA (SW480shRNA and SW680shRNA), and recombinant human TGFβ1 probed with TGF β1 antibody (n=3). D . Western blot of total TGFβ1 expression in whole cell lysate and in ECM fractions of CRCs wt RKO (treated with DMSO control) and wt SW480 cells folllowing treatment with TG2 cell permeable inhibitor 1-155 (1 μM). E . Cell surface TG2 activity measured by incorporation of biotin-X-cadavarine in RKO EV control cells, RKOTG2 cells, SW480 EV control cells and SW480shRNA cells measured as described in the Materials and Methods. Data are represented as mean ± S.D., (n=2), with each independent experiment performed in triplicate (* and #, p<0.05).

Article Snippet: CRCs were also treated with recombinant human (rh) TGFβ1 (2.5 ng/ml) (R&D Systems, Minneapolis, UK) or mouse anti-human TGFβ neutralising antibody (R&D Systems, Minneapolis, UK).

Techniques: Western Blot, Expressing, Control, Transduction, shRNA, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant, Activity Assay

Journal: Oncotarget

Article Title: Tissue transglutaminase induces Epithelial-Mesenchymal-Transition and the acquisition of stem cell like characteristics in colorectal cancer cells

doi: 10.18632/oncotarget.15370

Figure Lengend Snippet: A . Western blotting of CRCs showing presence of TGFβ receptor (TGFβR) I and TGFβRII in wt RKO, SW480 and SW620 whole cell lysates. B . Western blotting of whole cell lysates of wt CRCs RKO, SW480 and SW620. Cells showing presence or absence of EMT markers following TGFβ1 treatment with and without treatment with TGFβ neutralising antibody. C . Representative images of cell morphology of CRCs wt RKO and SW480 treated with rhTGFβ1 (2.5 ng/ml) and rhTGFβ1 plus TGFβ1 neutralising antibody PAN (20μg/ml). Equal numbers of cells were seeded and 48 h after treatment images were taken at 20× objective using a phrase contrast microscope. D . Western blot of whole cell lysates from wt RKO, SW480 and SW620 cells, showing expression of TG2 after TGFβ1 treatment, with or without treatment with TGFβ neutralising antibody. CRCs were treated with human recombinant TGFβ1 (2.5 ng/ml) and TGFβ neutralising antibody (NA) (20 μg/ml) for 48 h.

Article Snippet: CRCs were also treated with recombinant human (rh) TGFβ1 (2.5 ng/ml) (R&D Systems, Minneapolis, UK) or mouse anti-human TGFβ neutralising antibody (R&D Systems, Minneapolis, UK).

Techniques: Western Blot, Microscopy, Expressing, Recombinant

Journal: Oncotarget

Article Title: Tissue transglutaminase induces Epithelial-Mesenchymal-Transition and the acquisition of stem cell like characteristics in colorectal cancer cells

doi: 10.18632/oncotarget.15370

Figure Lengend Snippet: A . Western blotting showing ERK1/2 activation, after TGFβ1 (5.0 ng/ml) treatment over a time course of 2 h in wt RKO and SW480 cells. B . Western bloting of whole cell lysates of wt RKO and SW480 cells, showing expression of TG2 and ERK1/2 after treatment with TGFβ1 with or without ERK inhibitor PD98059 (10 μM). C . Western blotting of whole cell lysates from wt RKO and SW480 cells showing expression of ERK1/2, C-Jun and TGFβ1 with or without treatment of cells with ERK inhibitor PD98059 (10 μM). D . Western blotting of whole cell lysates of RKO and SW480 control cells and cells transduced with TG2 (RKOTG) or shRNA (SW480shRNA), showing TG2 expression, ERK1/2 activation and TGFβ1 expression. E . Western blotting of whole cell lysates showing activation of ERK1/2 in wt RKO and SW480 cells with or without treatment with TG2-sepcific cell permeable inhibitor 1-155 inhibitor (1 μM).

Article Snippet: CRCs were also treated with recombinant human (rh) TGFβ1 (2.5 ng/ml) (R&D Systems, Minneapolis, UK) or mouse anti-human TGFβ neutralising antibody (R&D Systems, Minneapolis, UK).

Techniques: Western Blot, Activation Assay, Expressing, Control, Transduction, shRNA