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. 2006 Sep 18;26(23):9126–9135. doi: 10.1128/MCB.00679-06

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FIG.3. — (A) RU486 abrogates dexamethasone induction of TTP. (Upper panel) Real-time RT-PCR analysis of A549 cells treated for 2 h with 1 μM RU486 (or vehicle) and/or 100 nM dexamethasone. Values are the means ± standard errors of the means for three experiments. *, P < 0.001. (Lower panel) Western analysis of A549 cells treated for 8 h with 10 μM RU486 (or vehicle) and/or dexamethasone (100 nM) as indicated. (B) Dexamethasone induction of TTP mRNA is abrogated by actinomycin D but not by cycloheximide. Representative results of experiments of real-time RT-PCR (three wells per treatment), run in duplicate, are shown. Subsequent experiments gave similar results. (Left panel) Real-time RT-PCR analysis of A549 cells pretreated with vehicle or actinomycin D (ACT.D, 10 μg/ml) for 30 min and then treated for 2 h with 100 nM dexamethasone as indicated. *, P < 0.001 (relative to control). (Right panel) Real-time RT-PCR analysis of A549 cells pretreated with vehicle or cycloheximide (CHX, 35 μM) for 1 h and then treated for 2 h with 100 nM dexamethasone as indicated. *, P < 0.001 (relative to control or versus CHX). (C) Dexamethasone enhances hTTP transcription. Nuclear run-on transcription analysis of A549 cells treated for 2 h with or without dexamethasone (100 nM) is shown. The value obtained for TTP mRNA is normalized to each internal control (18S or actin) and is depicted as the level relative to treated control cells. The results represent the mean values (± standard deviations) from two independent experiments, each run in duplicate. (D) GR occupancy of the TTP promoter and 3′ flanking region. In the ChIP assay, A549 cells were treated for 1 h with 1 μM dexamethasone, after which they were lysed, sonicated, and immunoprecipitated with either nonspecific (NS) antibody to rabbit immunoglobulin G or specific GR antibody. The immunoprecipitated 5′ flanking region (top gel) and 3′ flanking region of TTP (center gel) were identified by PCR. (Lower gel) Serving as a negative control, an intronic region of TTP was also analyzed with the same DNA as that in the experiments described above. Shown are representative results of three experiments.

FIG.3. — (A) RU486 abrogates dexamethasone induction of TTP. (Upper panel) Real-time RT-PCR analysis of A549 cells treated for 2 h with 1 μM RU486 (or vehicle) and/or 100 nM dexamethasone. Values are the means ± standard errors of the means for three experiments. *, P < 0.001. (Lower panel) Western analysis of A549 cells treated for 8 h with 10 μM RU486 (or vehicle) and/or dexamethasone (100 nM) as indicated. (B) Dexamethasone induction of TTP mRNA is abrogated by actinomycin D but not by cycloheximide. Representative results of experiments of real-time RT-PCR (three wells per treatment), run in duplicate, are shown. Subsequent experiments gave similar results. (Left panel) Real-time RT-PCR analysis of A549 cells pretreated with vehicle or actinomycin D (ACT.D, 10 μg/ml) for 30 min and then treated for 2 h with 100 nM dexamethasone as indicated. *, P < 0.001 (relative to control). (Right panel) Real-time RT-PCR analysis of A549 cells pretreated with vehicle or cycloheximide (CHX, 35 μM) for 1 h and then treated for 2 h with 100 nM dexamethasone as indicated. *, P < 0.001 (relative to control or versus CHX). (C) Dexamethasone enhances hTTP transcription. Nuclear run-on transcription analysis of A549 cells treated for 2 h with or without dexamethasone (100 nM) is shown. The value obtained for TTP mRNA is normalized to each internal control (18S or actin) and is depicted as the level relative to treated control cells. The results represent the mean values (± standard deviations) from two independent experiments, each run in duplicate. (D) GR occupancy of the TTP promoter and 3′ flanking region. In the ChIP assay, A549 cells were treated for 1 h with 1 μM dexamethasone, after which they were lysed, sonicated, and immunoprecipitated with either nonspecific (NS) antibody to rabbit immunoglobulin G or specific GR antibody. The immunoprecipitated 5′ flanking region (top gel) and 3′ flanking region of TTP (center gel) were identified by PCR. (Lower gel) Serving as a negative control, an intronic region of TTP was also analyzed with the same DNA as that in the experiments described above. Shown are representative results of three experiments.