18 research outputs found

    Concentrations of fecal corticosterone metabolites (FCM; mean ± SD) of female C57BL/6N and Crl:CD1 mice over a seven day period after separation.

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    <p>Preliminary study (n = 4; each strain). *, p≤0.05 for paired samples t-tests between day 1 and days 4, 5 and 6.</p

    Mean concentrations of fecal corticosterone metabolites (FCM) per mouse and lifetime, pooled for the corresponding seasonal sampling points.

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    <p>Mean concentrations of fecal corticosterone metabolites (FCM) per mouse and lifetime, pooled for the corresponding seasonal sampling points.</p

    Overview of maximum ages and sampling points for individual female mice of the C57BL/6N and Crl:CD1 strain mice in main study.

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    <p>Overview of maximum ages and sampling points for individual female mice of the C57BL/6N and Crl:CD1 strain mice in main study.</p

    Diurnal rhythmicity calculated separately per mouse and sampling point as coefficient of variation (CV).

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    <p>A linear regression analysis describes the slope of circadian oscillation of FCM concentrations over lifetime.</p

    Inducible, Dose-Adjustable and Time-Restricted Reconstitution of <i>Stat1</i> Deficiency <i>In Vivo</i>

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    <div><p>Signal transducer and activator of transcription (STAT) 1 is a key player in interferon (IFN) signaling, essential in mediating host defense against viruses and other pathogens. STAT1 levels are tightly regulated and loss- or gain-of-function mutations in mice and men lead to severe diseases. We have generated a doxycycline (dox) -inducible, FLAG-tagged <i>Stat1</i> expression system in mice lacking endogenous STAT1 (i.e. <i>Stat1<sup>ind</sup></i> mice). We show that STAT1 expression depends on the time and dose of dox treatment in primary cells and a variety of organs isolated from <i>Stat1<sup>ind</sup></i> mice. In bone marrow-derived macrophages, a fraction of the amount of STAT1 present in WT cells is sufficient for full expression of IFN-induced genes. Dox-induced STAT1 established protection against virus infections in primary cells and mice. The availability of the <i>Stat1<sup>ind</sup></i> mouse model will enable an examination of the consequences of variable amounts of STAT1. The model will also permit the study of STAT1 dose-dependent and reversible functions as well as of STAT1's contributions to the development, progression and resolution of disease.</p></div

    IFN-induced transcription of <i>Mx1</i> and <i>Irf1</i> at low or WT cellular amounts of STAT1<sup>FLAG</sup> and antiviral activity of STAT1<sup>FLAG</sup>.

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    <p><b>A</b>) BMMΦs of various genotypes were pretreated with 0.25 µg/ml dox for 24 h and stimulated with 100 U/ml IFNβ or IFNγ for 4 h or left untreated (w/o). RNA was isolated and cDNA used to analyze expression of <i>Stat1</i> (upper panel), <i>Mx1</i> (middle panel) and <i>Irf1</i> (lower panel) mRNA. <i>Ube2d2</i> was used for normalization and expression values were calculated relative to untreated WT cells. Results are shown as mean values ± SE from three independent experiments. For clarity p-values (*** p<0.001) are indicated only for differences between <i>Stat1<sup>ind</sup></i> cells pretreated with or without dox. Differences between <i>Stat1<sup>−/−</sup></i> and untreated <i>Stat1<sup>ind</sup></i> cells were significant (p<0.001) compared to WT and <i>Stat1<sup>ind</sup></i> cells pretreated with dox for all mRNAs analyzed except for basal <i>Irf1</i> expression. <b>B</b>) PEFs were isolated from WT, <i>Stat1<sup>−/−</sup></i> (<i>S1<sup>−/−</sup></i>) and <i>Stat1<sup>ind</sup></i> mice. 5×10<sup>3</sup> cells were plated onto each well of a 96-well plate, pretreated with 0.025 µg/ml dox for 24 h, stimulated with serial 2-fold dilutions of IFNγ starting at 100 U/ml for 24 h and infected with VSV at a moi of 0.5. After two or three days surviving cells were stained with crystal violet (left panel). Stained cells were solubilized and crystal violet intensity was measured at 595 nm. Resulting values were calculated relative to each untreated genotype. Mean values ± SE from three independent experiments are shown (right panel). <b>C–E</b>) BMMΦs were isolated from WT and <i>Stat1<sup>ind</sup></i> mice, pretreated with increasing amounts of dox (0.1, 0.125, 0.15, 0.25 and 10 µg/ml) for 24 h and stimulated with 100 U/ml IFNβ or IFNγ for 4 h or left untreated. RNA was isolated and cDNA used to analyze expression of <i>Stat1</i> (C), <i>Mx1</i> (D) and <i>Irf1</i> (E) mRNA as described in (A).</p

    STAT1<sup>FLAG</sup>-mediated defense against VSV and EMCV infections.

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    <p>WT and <i>Stat1<sup>ind</sup></i> mice were pretreated with the indicated concentrations of dox in the drinking water for one week or left untreated <b>A</b>) Mice were injected with 10<sup>5</sup> pfu/mouse VSV intraveniously (i.v.) and survival monitored for two weeks. Numbers of mice are indicated and results are shown from four (left panel) or two (right panel) independent experiments. There was no significant difference in survival between WT mice treated with or without dox. The survival of <i>Stat1<sup>ind</sup></i> mice with or without dox was significantly different (p<0.0002) from that of all other groups. <b>B</b>) Mice were injected with 10<sup>5</sup> pfu/mouse VSV intranasally (i.n.) and survival monitored for two weeks. Results are shown from three independent experiments. The survival rate of <i>Stat1<sup>ind</sup></i> mice without dox and <i>Stat1<sup>ind</sup></i> mice with dox was significantly different (p<0.0001) from that of all other groups. <b>C</b>) Mice were injected with 5 pfu/mouse EMCV intraperitoneally (i.p.) and survival monitored for two weeks. Results are shown from two independent experiments. The survival of <i>Stat1<sup>ind</sup></i> mice without dox was significantly different (p<0.0024) from that of all other groups.</p

    Phosphorylation and DNA binding of IFN-activated STAT1<sup>FLAG</sup> in BMMΦs.

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    <p>BMMΦs were isolated from WT and <i>Stat1<sup>ind</sup></i> mice. Cells were pretreated with 0.25 μg/ml dox for 24 h and stimulated with 100 U/ml IFNβ or IFNγ for 15 or 60 min or left untreated. Results are shown from one representative out of three independent experiments. <b>A</b>) Protein lysates were subjected to Western blot analysis for tyrosine 701-phosphorylated STAT1 (P-Tyr-STAT1; upper panel) or serine 727-phosphorylated STAT1 (P-Ser-STAT1; lower panel). Membranes were reprobed with antibodies against STAT1 and FLAG. Loading was controlled by reprobing the membranes with β-tubulin. <b>B</b>) EMSAs were performed to analyze binding of ISGF3 to ISRE (upper panel) or STAT1/STAT1 to GAS elements (lower panel).</p

    Anemic phenotype is reversible upon withdrawal of temporary ILEI overexpression.

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    (A, B) Mean (A) body weight and (B) HGB concentration (top left), RBC count (middle left), HCT percentage (bottom left), MCV (top right), MCH (middle right) and MCHC (bottom right) ± SEM of R26-ILEIind mice kept temporarily on Dox water from week 3 to 13 of age and measured weekly until week 15 of age compared to littermates used as Dox treatment and genetic controls. (A, B) Statistical significance was determined by one-way ANOVA and is marked with asterisks (*p.05; **p.01; ***p.001; ****p.0001).</p
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