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Results: 8

1.
Fig. 1.

Fig. 1. From: Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus.

GR and ERα expression in response to Dex and E2 treatment in ECC1 cells. A, Expression of GR and ERα was measured by QRT-PCR. ECC1 cells treated with 100 nm Dex, 10 nm E2, or 100 nm Dex and 10 nm E2 were normalized to the housekeeping gene Cyclophilin B and compared with vehicle-treated cells. Bar graphs show mean ± sem. *, P < 0.05 as determined by ANOVA. B, GR and ERα protein levels were measured by Western blotting in whole-cell lysates from ECC1 cells treated for 6 and 24 h with vehicle (Veh), 100 nm Dex, 10 nm E2, or 100 nm Dex and 10 nm E2 (D + E2). The housekeeping gene β-actin was used as a control. The figure is representative of three experiments.

Shannon Whirledge, et al. Endocrinology. 2013 January;154(1):499-510.
2.
Fig. 6.

Fig. 6. From: Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus.

siRNA-mediated knockdown of ERα inhibits E2-mediated repression of GILZ expression. A, ECC1 cells, transfected with ERα siRNA or NTC, were assessed for the extent of knockdown compared with NTC by QRT-PCR and Western blotting. GR expression levels were evaluated by QRT-PCR under in ERα knockdown ECC1 cells. Values were normalized to the housekeeping genes Cyclophilin B (QRT-PCR) or β-actin (Western blotting). Bar graphs show mean ± sem. *, P < 0.05 as determined by ANOVA. B, ECC1 cells transfected with NTC or ERα siRNA were treated for 6 h with vehicle (Veh), 100 nm Dex, 10 nm E2, or 100 nm Dex and 10 nm E2 (D + E2). Expression of GILZ was evaluated by QRT-PCR. Values were normalized to the housekeeping gene Cyclophilin B and compared with vehicle-treated cells. Bar graphs show mean ± sem. The symbols represent values that are statistically different at P < 0.05 from one another as determined by Tukey's post hoc analysis after ANOVA.

Shannon Whirledge, et al. Endocrinology. 2013 January;154(1):499-510.
3.
Fig. 8.

Fig. 8. From: Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus.

Global E2-mediated repression of GILZ expression in the mouse uterus. A, Proportion uterine weight relative to body weight was determined in intact and OVX mice (n = 7 per group) after 4 h of treatment with 1 mg/kg of Dex, 10 μg/kg of E2, or 1 mg/kg of Dex and 10 μg/kg of E2. Bar graphs show mean ± sem. *, P < 0.05 as determined by ANOVA. B, GILZ expression (evaluated by QRT-PCR and Western blotting) was determined in intact and OVX mice when stimulated with vehicle (Veh), 1 mg/kg of Dex, 10 μg/kg of E2, or 1 mg/kg of Dex and 10 μg/kg of E2 (D + E2). mRNA was normalized to Cyclophillin B, and protein was normalized to β-actin. Bar graphs show mean ± sem. **, P < 0.01 as determined by ANOVA. C, Proportion uterine weight relative to body weight was determined in mice (n = 7 per group) after 24 h of treatment with 1 mg/kg of Dex, 10 μg/kg of E2, or 1 mg/kg of Dex and 10 μg/kg of E2. Bar graphs show mean ± sem. The symbols represent values that are statistically different at P < 0.05 from one another as determined by Tukey's post hoc analysis after ANOVA. D, Western blot analysis determined GILZ expression in whole uterus from intact and OVX mice. Values were normalized to the house keeping protein β-actin. Bar graphs show mean ± sem. *, P < 0.05 as determined by ANOVA.

Shannon Whirledge, et al. Endocrinology. 2013 January;154(1):499-510.
4.
Fig. 7.

Fig. 7. From: Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus.

Association of GR and ERα with the promoter of GILZ. A, Recruitment of GR and ERα to GRE at position −1919 to −1794 was assessed by ChIP assay after treatment with vehicle (Veh), 100 nm Dex, 10 nm E2, or 100 nm Dex and 10 nm E2 (D + E2) for 1 h in ECC1 cells. Enrichment of the promoter region measured by QRT-PCR. Bar graphs show mean ± sem. *, P < 0.05 as determined by ANOVA. B, Recruitment of GR and ERα to the GILZ TSS was examined by ChIP after treatment with vehicle, 100 nm Dex, 10 nm E2, or 100 nm Dex and 10 nm E2. Enrichment measured by primers directed to the TSS and quantified by QRT-PCR. Bar graphs show mean ± sem. *, P < 0.05 as determined by ANOVA. C, Recruitment of activated Pol II to the TSS of GILZ determined by ChIP after treatment of ECC1 cells with vehicle, 100 nm Dex, 10 nm E2, or 100 nm Dex and 10 nm E2 for 1 h. Enrichment measured by QRT-PCR. Bar graphs show mean ± sem. The symbols represent values that are statistically different at P < 0.01 from one another as determined by Tukey's post hoc analysis after ANOVA. D, Recruitment of GR to a NFκB site in IL-8 and ERα to an ERE in TFF1 were analyzed by ChIP and quantified by QRT-PCR for control. Bar graphs show mean ± sem. **, P < 0.01 as determined by ANOVA.

Shannon Whirledge, et al. Endocrinology. 2013 January;154(1):499-510.
5.
Fig. 2.

Fig. 2. From: Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus.

Glucocorticoid-responsive GILZ expression in ECC1. A, Expression of GILZ was measured by QRT-PCR in ECC1 cells treated for 6 or 24 h with 100 nm Dex, 10 nm E2, or 100 nm Dex and 10 nm E2 (D + E2). Values were normalized to the housekeeping gene Cyclophilin B and compared with vehicle-treated (Veh) cells. Bar graphs show mean ± sem. The symbols represent values that are statistically different at P < 0.05 from one another as determined by Tukey's post hoc analysis after ANOVA. Treatment groups without symbols are not statistically different from each other. B, Whole-cell lysates were subjected to Western blot analysis for GILZ. The housekeeping gene β-actin was used as a control and for normalization. The bar graph represents the quantification of four experiments. C, Expression of MKP1 and FKBP5 was measured by QRT-PCR in ECC1 cells treated for 6 h with 100 nm Dex, 10 nm E2, or 100 nm Dex and 10 nm E2. Values were normalized to the housekeeping gene Cyclophilin B and compared with vehicle-treated cells. Bar graphs show mean ± sem. The symbols represent values that are statistically different at P < 0.05 from one another as determined by Tukey's post hoc analysis after ANOVA.

Shannon Whirledge, et al. Endocrinology. 2013 January;154(1):499-510.
6.
Fig. 5.

Fig. 5. From: Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus.

ER depletion by ICI 182,780 inhibits E2-mediated repression of GILZ expression. A, Whole-cell lysates from ECC1 cells treated over a time course with 1 or 10 μm ICI 182,780 (ICI) were subjected to Western blot analysis for ERα. The housekeeping gene β-actin was used as a control and for normalization. The bar graph shows mean ± sem and represents the quantification of four experiments. *, P < 0.05 as determined by ANOVA. B, GILZ expression was determined by QRT-PCR in ECC1 cells cotreated with vehicle (Veh), 1 μm, or 10 μm ICI and 24 h of vehicle, 100 nm Dex, 10 nm E2, or 100 nm Dex and 10 nm E2 (D + E2) for 24 h. Values were normalized to the housekeeping gene Cyclophilin B. Bar graphs show mean ± sem. Symbols represent groups with means statistically different at P < 0.05 as determined by ANOVA. C, Whole-cell lysates from ECC1 cells treated with or without 1 μm ICI and vehicle, 100 nm Dex, 10 nm E2, or 100 nm Dex and 10 nm E2 for 24 h were subjected to Western blot analysis for GILZ. The housekeeping gene β-actin was used as a control and for normalization. The bar graph shows mean ± sem and represents the quantification of four experiments. Symbols represent groups with means statistically different at P < 0.05 as determined by ANOVA.

Shannon Whirledge, et al. Endocrinology. 2013 January;154(1):499-510.
7.
Fig. 3.

Fig. 3. From: Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus.

Kinetics of GILZ regulation in response to Dex and E2 treatment. A, Expression of GILZ was measured by QRT-PCR in ECC1 cells treated for 0, 0.5, 1, 2, 4, 6, and 24 h with 100 nm Dex or 100 nm Dex and 10 nm E2 (D + E2). Graph shows mean ± sem. *, P < 0.05 as determined by ANOVA. B, Expression of GILZ was measured by QRT-PCR in ECC1 cells treated with vehicle (Veh) or 100 nm Dex for 6 h with varying lengths of time with 10 nm E2 (0, 6, 4, 2, and 1 h). Values were normalized to the housekeeping gene Cyclophilin B. Bar graphs show mean ± sem. The symbols represent values that are statistically different at P < 0.05 from one another as determined by Tukey's post hoc analysis after ANOVA. C, Expression of GILZ was measured by QRT-PCR in ECC1 cells treated with vehicle, Dex at varying concentrations, or Dex at varying concentrations with 10 nm E2. Graph shows mean ± sem. *, P < 0.05 as determined by ANOVA. D, Expression of GILZ was measured by QRT-PCR in ECC1 cells treated with vehicle, E2 at varying concentrations, or 100 nm Dex with varying concentrations of E2. Graph shows mean ± sem. *, P < 0.05 as determined by ANOVA.

Shannon Whirledge, et al. Endocrinology. 2013 January;154(1):499-510.
8.
Fig. 4.

Fig. 4. From: Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus.

Transcriptional regulation is a likely mechanism of E2-mediated GILZ antagonism. A, ECC1 cells were treated with 10 μm cycloheximide for 1 h before hormone treatment. Cells were then administered 100 nm Dex, 10 nm E2, or 100 nm Dex and 10 nm E2 (D + E2) for 6 h and GILZ mRNA evaluated by QRT-PCR was compared to vehicle (Veh). Values were normalized to the housekeeping gene Cyclophilin B. Bar graphs show mean ± sem. The symbols represent values that are statistically different at P < 0.05 from one another as determined by Tukey's post hoc analysis after ANOVA. B, RNA decay was measured by treating ECC1 cells with vehicle, 100 nm Dex, or 100 nm Dex and 10 nm E2 for 4 h to induce gene expression followed by the addition of 5 μg/μl Actinomycin D. GILZ mRNA was measured by QRT-PCR at time points 0, 2, 4, and 16 h after the addition of Actinomycin D. Amount of mRNA at time 0 was set to 100%, and subsequent time points are reported as a percentage of 100. C, Relative levels of nascent RNA was measured by QRT-PCR in ECC1 cells after treatment with vehicle, 100 nm Dex, or 100 nm Dex and 10 nm E2 for 0, 2, 4, and 6 h. Values were normalized to nascent RNA for the housekeeping gene Cyclophilin B. Graph shows mean ± sem. *, P < 0.05 as determined by ANOVA.

Shannon Whirledge, et al. Endocrinology. 2013 January;154(1):499-510.

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