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21733716[PMID] - PMC - NCBI

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1.
Fig 2

Fig 2. From: MKP-1 Regulates Cytokine mRNA Stability Through Selectively Modulation Subcellular Translocation of AUF1.

MKP-1 specifically attenuated IL-6, TNF-α and IL-10 cytokine expression. Rat macrophages were transduced with Ad.MKP-1 or control Ad.LacZ (300 MOI) for 48 h.Primary bone marrow macrophages were either harvested from MKP-1 KO mice or WT mice. Rat macrophages and bone marrow macrophages from mice were either unstimulated or stimulated with 100ng/ml of LPS (from Escherichia coli 0127:B8) for 24 h. (a) IL-6 expression in rat macrophages (n=5, *p<0.05). (b) TNF- α expression in rat macrophages (n=5, ***p<0.001). (c)IL-10 expression in rat macrophages (n=5, ***p<0.001). (d) IL-6 expression in mouse macrophages (n=3, ***p<0.001). (e) TNF-α expression in mouse macrophages (n=3, **p<0.01). (f) IL-10 expression in mouse macrophages (n=3, **p<0.01). These data are representative of three separate experiments.

Hong Yu, et al. Cytokine. ;56(2):245-255.
2.
Fig 1

Fig 1. From: MKP-1 Regulates Cytokine mRNA Stability Through Selectively Modulation Subcellular Translocation of AUF1.

Ad.MKP-1 specifically dephosphorylated MAPKs in rat macrophages after LPS stimulation. Rat macrophages were transduced with either Ad.MKP-1, or control Ad.LacZ (300 MOI), or treated with HEPES buffered saline for 48 h. Cells were either unstimulated or stimulated with 1μg/ml of LPS (from Escherichia coli 0127:B8) for indicated time periods. MKP-1, p-JNK (p54, p46), p-p38, p-ERK (p44, p42), p-38 MAPK, and p-NF-κB p65 protein expressions were evaluated by Western blot assay. Total p-38 MAPK served as a loading control.

Hong Yu, et al. Cytokine. ;56(2):245-255.
3.
Fig 6

Fig 6. From: MKP-1 Regulates Cytokine mRNA Stability Through Selectively Modulation Subcellular Translocation of AUF1.

MKP-1 modulates subcellulartranslocalization of AUF1.Primary bone marrow macrophages were either harvested from MKP-1 KO mice or WT mice. Cells were either unstimulated or stimulated with 1μg/ml of LPS (from Escherichia coli 0127:B8) for 4 h. The cells were stained AUF1 by immunofluorescence staining. The nucleus was stained with DAPI. The cytoplasm was stained with Alexa fluorphalloidin 568-labeled actin. For each experiment, each sample was duplicated.Digital images were recorded using confocal microscopy (Leica, Bannockburn, IL). Five fields (about 100 cells) from each sample were counted. Results are representative of three separate experiments.

Hong Yu, et al. Cytokine. ;56(2):245-255.
4.
Fig 3

Fig 3. From: MKP-1 Regulates Cytokine mRNA Stability Through Selectively Modulation Subcellular Translocation of AUF1.

MKP-1 inhibited IL-6, TNF-α, and IL-10 mRNA stimulated by LPS. Rat macrophages were transduced with Ad.MKP-1 or control Ad.LacZ (300 MOI) for 48 h. Primary bone marrow macrophages were either harvested from MKP-1 KO mice or WT mice. Cells were either unstimulated or stimulated with 100ng/ml of LPS (from Escherichia coli 0127:B8) for 4 and 8 h. (a)IL-6 mRNA expression in rat macrophages (n=4, ***p<0.001). (b)TNF-α mRNA expression in rat macrophages (n=4, ***p<0.001). (c) IL-10 mRNA expression in rat macrophages (n=4, ***p<0.001). (d) IL-6 mRNA expression in mouse bone marrow macrophages (n=3, *p<0.05, ***p<0.001). (e) TNF-α mRNA expression in mouse bone marrow macrophages (n=3, **P<0.01, ***p<0.001). (f) IL-10 mRNA expression in mouse bone marrow macrophages (n=3, ***p<0.001).These data are representative of three separate experiments.

Hong Yu, et al. Cytokine. ;56(2):245-255.
5.
Fig. 7

Fig. 7. From: MKP-1 Regulates Cytokine mRNA Stability Through Selectively Modulation Subcellular Translocation of AUF1.

AUF1 was associated with degradation of IL-6, TNF-α, and IL-10 mRNA. Bone marrow cells from MKP-1 WT mice were treated with either AccellSMARTpool AUF1 siRNA or control Accell Non-targeting Pool siRNA (500 nM) for 72 h. Cells were then stimulated with LPS (from Escherichia coli 0127:B8, 100 ng/ml) for 4 h. RNA decay assay was performed after addition of actinomycin D. (a) AUF1 mRNA expression (n=3, ***p<0.001). (b) IL-6 mRNA remaining percentage (n=3, *p<0.05, **p<0.01). (c)TNF-α mRNA remaining percentage (n=3, *p<0.05). (d) IL-10 mRNA remaining percentage (n=3, *p<0.05). These data are representative of three separate experiments.

Hong Yu, et al. Cytokine. ;56(2):245-255.
6.
Fig 4

Fig 4. From: MKP-1 Regulates Cytokine mRNA Stability Through Selectively Modulation Subcellular Translocation of AUF1.

MKP-1 increased the degradation of mRNAs of IL-6, TNF-α, and IL-10 stimulated by LPS. Rat macrophages were transduced with Ad.MKP-1 or control Ad.LacZ (300 MOI) for 48 h. Primary bone marrow macrophages were either harvested from MKP-1 KO mice or WT mice. Cells were stimulated with 100 ng/ml of LPS (from Escherichia coli 0127:B8) for 4 h. Actinomycin D (10μg/ml) was added to the cells to stop mRNA transcription and the total RNAs were harvested at various time points indicated in the graphs after actimomycin D treatment. (a)IL-6 mRNA remaining percentage in rat macrophages (n=3, *p<0.05, **p<0.01). (b)TNF-α mRNA remaining percentage in rat macrophages (n=3, **p<0.01, ***p<0.001).(c)IL-10 mRNA remaining percentage in rat macrophages (n=3, *p<0.05, **p<0.01). (d) IL-6 mRNA remaining percentage in mouse bone marrow macrophages (n=3, *p<0.05, **p<0.01). (e)TNF-α mRNA remaining percentage in mouse bone marrow macrophages (n=3, *p<0.05, **p<0.01).(f) IL-10 mRNA remaining percentage in mouse bone marrow macrophages (n=3, *p<0.05, **p<0.01). These data are representative of three separate experiments.

Hong Yu, et al. Cytokine. ;56(2):245-255.
7.
Fig 8

Fig 8. From: MKP-1 Regulates Cytokine mRNA Stability Through Selectively Modulation Subcellular Translocation of AUF1.

AUF1 subcellular translocation post LPS stimulation in MKP-1 WT cells was associated with enhanced binding with IL-6, TNF-α, and IL-10 mRNAs. Primary bone marrow macrophages were either harvested from MKP-1 KO mice or WT mice. Cells were either unstimulated or stimulated with 1μg/ml of LPS (from Escherichia coli 0127:B8) for 4 h. Protein A agarose beads coated with either anti-AUF1 or control normal rabbit serum were immunoprecipitated (IP) with cell lysates from bone marrow cells with or without LPS stimulation. RNA was extracted from IP products and RT-qPCR was performed. (a) IL-6 mRNA expression (n=3, *p<0.05) (b) TNF-α mRNA expression (n=3, **p<0.01). (c) IL-10 mRNA expression. (n=3, *p<0.05). IL-6, TNF-α, and IL-10 mRNA were normalized to 10% input RNA endogenous beta-actin expression. The displayed result is representative of three separate experiments.

Hong Yu, et al. Cytokine. ;56(2):245-255.
8.
Fig 5

Fig 5. From: MKP-1 Regulates Cytokine mRNA Stability Through Selectively Modulation Subcellular Translocation of AUF1.

MKP-1 modulates subcellulartranslocalization of AUF1. Rat macrophages were transduced with Ad.MKP-1 or control Ad.LacZ (300 MOI), or treated with HEPES buffered saline for 48 h. Primary bone marrow macrophages were either harvested from MKP-1 KO mice or WT mice. The macrophages were either unstimulated or stimulated with 1μg/ml of LPS (from Escherichia coli 0127:B8) for 2 or 4 h. The nuclear and cytoplasmic proteins were extracted and analyzed by Western blot assay. Cytoplasmic protein (30 μg) and nuclear protein (15 μg) was loaded on the gel. (a) MKP-1, p-JNK (p54, p46), p-p38, p-ERK (p44, p42), AUF1 (p45, p42, p40, p37), and TTP proteins expression in the cytoplasm vs. in the nucleus of rat macrophages. (b) AUF1 (p45, p42, p40, p37), and TTP proteins expression in the cytoplasm vs. in the nucleus of mouse bone marrow macrophages.The GAPDH (cytoplasmic protein) and Lamin A/C (nuclear protein) are loading controls for cytoplasmic protein and nuclear protein separately. (c) AUF1 and TTP protein density in rat macrophages transduced with AdMKP-1 (black bars), or Ad.LacZ (white bars) or treated with HEPES buffered saline (gray bars) (n=3, *p<0.05). (d) AUF1 and TTP protein density in bone marrow (BM) macrophages from MKP-1 KO mice (black bars) and WT mice (white bars)(n=3, *p<0.05). The AUF1 and TTP protein density/mm2 was analyzed by Quantity One software and normalized by cytoplasmic protein GAPDH or nuclear protein Lamin A/C respectively. These data are representative of three separate experiments.

Hong Yu, et al. Cytokine. ;56(2):245-255.

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