Journal: PLoS ONE

Article Title: Tissue Inhibitor of Metalloproteinase 1 Is Preferentially Expressed in Th1 and Th17 T-Helper Cell Subsets and Is a Direct Stat Target Gene

doi: 10.1371/journal.pone.0059367

Figure Lengend Snippet: ( A ) Naïve CD4 + T cells were stimulated for three days in media alone, iTreg conditions or Th17(β) conditions. Subsequently, mRNA was isolated and Il17a and Timp1 mRNA analysed by quantitative-PCR. ( B ) Naïve CD4 + T cells were stimulated in media alone, IL-6 alone, iTreg conditions, Th17(β) conditions or Th17(23) conditions. After three days, secreted TIMP1 was measured by ELISA. ( C ) Naïve CD4 + T cells were polyclonally stimulated in media alone (Th0) or in either Th17(β) conditions or Th17(23) conditions in the presence or absence of IL-1β. After three days, secreted TIMP1 was measured by ELISA. ( D ) Naïve CD4 + T cells were stimulated in media alone or under Th1 conditions, Th2 conditions or Th17(β) conditions. After three days, secreted TIMP1 was measured by ELISA. Histograms represent mean values (n = 3 per group), error bars represent s.d. Data are representative of two independent experiments.

Article Snippet: Cytokine and secreted protein production in cell culture supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA) with mouse IL-17 Quantikine assay kits (R&D Systems, Minneapolis, MN) and TIMP1 Quantikine assay kits (R&D Systems) according to the manufacturer’s instructions.

Techniques: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Tissue Inhibitor of Metalloproteinase 1 Is Preferentially Expressed in Th1 and Th17 T-Helper Cell Subsets and Is a Direct Stat Target Gene

doi: 10.1371/journal.pone.0059367

Figure Lengend Snippet: Naïve CD4 + wild type B6 T cells ( A ) or Timp1 −/− T cells ( B ) were stimulated either under Th0 (media alone), IL-6 alone, Th1 conditions, Th2 conditions, iTreg conditions, Th17(β) or Th17(23) conditions. After three days, samples of supernatant were run on a protein gel together with control samples of TIMP1 and TIMP2. TIMP activity was determined by the ability of the protein gel to resist digestion by metaloprotease. Naïve CD4 + wild type B6 T cells ( C ) or Timp1 −/− T cells ( D ) were stimulated either under Th0 (media alone), IL-6 alone, Th1 conditions, Th2 conditions, iTreg conditions, Th17(β) or Th17(23) conditions. After three days, samples of supernatant were run on a protein gel together with control samples of MMP9. MMP9 activity was determined by its ability to digest protein within the gel. Results are representative of two independent experiments.

Article Snippet: Cytokine and secreted protein production in cell culture supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA) with mouse IL-17 Quantikine assay kits (R&D Systems, Minneapolis, MN) and TIMP1 Quantikine assay kits (R&D Systems) according to the manufacturer’s instructions.

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Tissue Inhibitor of Metalloproteinase 1 Is Preferentially Expressed in Th1 and Th17 T-Helper Cell Subsets and Is a Direct Stat Target Gene

doi: 10.1371/journal.pone.0059367

Figure Lengend Snippet: ( A ) Naïve CD4 + T cells from Stat3 fl/fl (black bars) or Stat3 fl/fl ;CD4-Cre (white bars) mice were stimulated in media alone (Th0), Th1 conditions or Th17(β) conditions. After three days, secreted TIMP1 was measured by ELISA. ( B ) Naïve CD4 + T cells from wild type (black bars) or Stat4 −/− (grey bars) were stimulated in media alone (Th0), Th1 conditions or Th17(β) conditions. After three days secreted TIMP1 was measured by ELISA. Histograms represent mean values (n = 3 per group), error bars represent s.d. Data are representative of two independent experiments.

Article Snippet: Cytokine and secreted protein production in cell culture supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA) with mouse IL-17 Quantikine assay kits (R&D Systems, Minneapolis, MN) and TIMP1 Quantikine assay kits (R&D Systems) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Tissue Inhibitor of Metalloproteinase 1 Is Preferentially Expressed in Th1 and Th17 T-Helper Cell Subsets and Is a Direct Stat Target Gene

doi: 10.1371/journal.pone.0059367

Figure Lengend Snippet: Chromatin signatures as defined by the presence of H3K4Me3 or STAT transcription factor binding at the Timp1 gene locus, illustrated at the top of the figure. Th17(β) polarized cells were re-stimulated with IL-6 and immunoprecipitated with anti-H3K4Me3 or STAT3 (upper two panels). The STAT3 dataset is taken from , the H3K4Me3 dataset is representative of two independent experiments . Th1 polarised cells were re-stimulated with CD3/CD28 and IL-12, immunoprecipitated with anti-STAT4 or with anti-STAT1 (lower two panels). The STAT4 dataset is taken from and the STAT1 dataset is taken from .

Article Snippet: Cytokine and secreted protein production in cell culture supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA) with mouse IL-17 Quantikine assay kits (R&D Systems, Minneapolis, MN) and TIMP1 Quantikine assay kits (R&D Systems) according to the manufacturer’s instructions.

Techniques: Binding Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Tissue Inhibitor of Metalloproteinase 1 Is Preferentially Expressed in Th1 and Th17 T-Helper Cell Subsets and Is a Direct Stat Target Gene

doi: 10.1371/journal.pone.0059367

Figure Lengend Snippet: Naïve CD4 + T cells from wild type ( A ) or Timp1 −/− ( B ) mice were stimulated under Th0, Th1 or Th17(β) conditions in the presence or absence of TIMP1. After three days, cells were fixed and assessed for IFN-γ and IL-17 expression. Naïve CD4 + T cells from wild type or Timp1 −/− mice were stimulated under Th1 ( C ) or Th17(β) ( D ) conditions for three days. The percentage IFN-γ + ( C ) or IL-17 + ( D ) cells were determined by intracellular staining. The histograms indicate mean ±s.e.m and the data were obtained from four independent experiments.

Article Snippet: Cytokine and secreted protein production in cell culture supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA) with mouse IL-17 Quantikine assay kits (R&D Systems, Minneapolis, MN) and TIMP1 Quantikine assay kits (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Tissue Inhibitor of Metalloproteinase 1 Is Preferentially Expressed in Th1 and Th17 T-Helper Cell Subsets and Is a Direct Stat Target Gene

doi: 10.1371/journal.pone.0059367

Figure Lengend Snippet: Naïve CD4 + T cells from 2D2 or Timp1 −/− 2D2 mice were stimulated under Th1 conditions ( A ), Th17(β) conditions ( B ) or Th17(23) conditions ( C ). After three days cells were fixed and assessed for IFN-γ and IL-17 expression (top panels). Next 10 6 polarized cells from each group were adoptively transferred into Rag2 −/− recipients, which were followed for signs of neurological disease. Data show mean ±s.e.m of the EAE clinical score of 5 mice per group. After 20 days (Mice receiving Th1 cells) or 35 days (Mice receiving Th17 cells), lymphocytes were isolated from the spinal chords from animals with a clinical score of 3.5 and IL-17 and IFN-γ expression were determined by intracellular staining (lower panels). Significance was determined by a Mann-Whitney U test, * P <0.05. Data are representative of two independent experiments.

Article Snippet: Cytokine and secreted protein production in cell culture supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA) with mouse IL-17 Quantikine assay kits (R&D Systems, Minneapolis, MN) and TIMP1 Quantikine assay kits (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Isolation, Staining, MANN-WHITNEY

Journal:

Article Title: Estrogen Decrease in Tight Junctional Resistance Involves Matrix-Metalloproteinase-7-Mediated Remodeling of Occludin

doi: 10.1210/en.2006-1120

Figure Lengend Snippet: A–E, Effects of treatment with bafilomycin A1. Estrogen-depleted or estrogen-treated cells were cotreated for 30 min with bafilomycin A1 (Baf-A1, 1 μM, added to the luminal solution). A, Total homogenates were fractionated by gel electrophoresis and immunoblotted with the anti-MMP7-F-12 antibody. Parallel fractions were immunoblotted with the anti-GAPDH antibody. B, Densitometry of the data in A (means ± SD of three experiments). Veh, vehicle. Ratios of MMP-7 21 kDa per GAPDH were normalized to an arbitrary unit (A.U.) of 10 for the SFM-Veh category in A. C, Conditioned media were obtained from the luminal compartment of estrogen-only-treated cells (Est) or Est + Baf-A1-treated cells. Samples were concentrated by centrifugation, and aliquots containing equal amounts of proteins were separated by gel electrophoresis and immunoblotted (IB) with the MMP-7 Nab antibody. D, Densitometry of the data in C (means ± SD of three experiments). Ratios of MMP-7 per total (T.) protein were normalized to an arbitrary unit (A.U.) of 10 for the 29-kDa-Est category in C. a and b, P < 0.01, compared with Est. E, Samples of conditioned media from C were analyzed by gelatin zymography. Experiments were repeated three times with similar trends. F, pH effects on MMP-7 activity in vitro. MMP-7 (± APMA) were dissolved in PBS (pH 7.4 or 6.0), separated by gel electrophoresis, and assayed by gelatin zymography. The experiments were repeated three times with similar trends. G, Levels of TIMP-1 and TIMP-2 (means ± SD of three experiments) in luminal conditioned media of estrogen-depleted and estrogen-treated cells were determined by ELISA. H, Conditioned media were collected from the luminal compartments of cultures containing estrogen-depleted or estrogen-treated hEVECs and mixed with 1 μg/ml anti-MMP-7 Nab antibody (to inactivate the MMP-7). Eight microliters of the samples were mixed with 2 μl of conditioned media collected from cultures of stromal cells previously treated with estrogen plus progesterone followed by withdrawal from that treatment (as in Fig. 4). The combined mixed samples were fractionated by gel electrophoresis and assayed by gelatin zymography. The experiments were repeated three times with similar trends.

Article Snippet: Commercially available ELISA kits were used to measure TIMP-1 (MTM100; R&D, Minneapolis MN) or TIMP-2 (RPN2618; Amersham, http://proteomics.amershambiosciences.com ) using 200–400 μ g of protein per assay according to suppliers’ protocols.

Techniques: Nucleic Acid Electrophoresis, Centrifugation, Zymography, Activity Assay, In Vitro, Enzyme-linked Immunosorbent Assay