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