28 research outputs found

    Human ENPP1 over-expression inhibits DMI-induced adipocyte maturation in 3T3-L1 cells.

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    <p>Human ENPP1 was over-expressed in 3T3-L1 using retrovirus transfection with CMV promoter. Panel A. ENPP1 protein content in cells containing vector only and in cells over-expressing human ENPP1 K121K or K121Q. ENPP1 was identified at 130 kDa using antibody from Imegex Corp. (San Diego, CA). Panel B. Oil-red-O stained 3T3-L1 cells after induction of adipogenesis. Cells were 100% confluent at the time of assay. Cellular triglyceride content is presented as fold changes from the triglyceride content of cells containing vector. Results are reported as mean and SD from measurements performed three times (8 wells measured in each experiment). Panel C. Cells were plated on 6-well plate and induced to differentiation with DMI. At different days, as indicated, the cell number was counted. Results are reported as mean and SD from measurements repeated three times (3 wells measured in each experiment). Open squares represent the data for the vector 3T3-L1. Open circles represent the data for 3T3-L1 over-expressing human ENPP1 K121K. Dark circles represent the data for 3T3-L1 over-expressing human ENPP1 K121Q. Cell death was measured at day 2 and day 4 of differentiation with DMI. Results are shown as mean and SD of percentage of dead cells relative to total cells. Measurements were performed for number of viable and dead cells in three wells for each cell type. These experiments were repeated 3 times. * p<0.05 for t-test between 3T3-L1 over-expressing human ENPP1 K121K and the vector group. † p<0.05 for t-test between 3T3-L1 over-expressing human ENPP1 K121Q and the vector group.</p

    Expression of ENPP1 decreases in the early maturation phase of 3T3-L1 into adipocytes.

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    <p>3T3-L1 cells were induced to differentiate into adipocytes by desamethasone, 3-iso-butyl-1-methylxanthine and insulin (DMI). The RNA was isolated at different time points, as indicated. Data show real time PCR for expression of ENPP1 and other genes involved in adipocyte maturation. Mean and SD from 3 experiments are presented in arbitrary units after correction for 18S expression. One-way ANOVA p-value for multiple group comparison was <0.05 for each of the gene expression data shown in figure. Pre-adipocyte are shown as dark bars, mature adipocyte are shown as open bars</p

    Human ENPP1 K121K and K121Q over-expression induce changes in the expression of genes present of 3T3-L1 induced to maturation.

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    <p>The expression levels of several adipogenic markers in vector only, in ENPP1 K121K and ENPP1 K121Q stable cell lines 10 days after DMI induction were measured by QPCR and normalized by the level of 18S. Experiments were repeated 3 times. The average values and SD are shown for the vector (open bars) and he transfected cells (dark bars).</p

    Kinetic expression of chemokines in rat vascular smooth muscle cells.

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    <p>Total RNA was extracted from rat smooth vascular muscle cells treated with IFN-γ, LPS, or IFN-γ plus LPS for different times as indicated and reverse transcribed into cDNA for measurement of CCL5 (A), IP-10 (B) and MCP-1 (C) expression by qRT-PCR. Data were normalized relative to GAPDH mRNA expression levels in each respective sample and further normalized to the results from the un-treated group, which was set as 1.</p

    The role of p38 MAPK in IP-10 expression.

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    <p>(A) 1×10<sup>6</sup> A7r5 cells were treated with different amounts of a p38 MAPK antagonist SB203580 for 1 h prior to addition of various stimuli as indicated. An identical amount of dissolvent DMSO was used for negative controls. 6 hrs later, total RNA was extracted for measurement of IP-10 mRNA expression by qRT-PCR. 5 µg of different expression plasmids encoding different molecules involved in the p38 MAPK signaling pathway including p38α (B), and DN-MK2 (C), as well as MLK3, MKK3, and MKK6 (D) or empty vector were transiently transfected with Lipofectamine. 48 h after transfection, the cells were treated with LPS (1 µg/ml) plus IFN-γ (10 ng/ml) for 6 h, followed by measurement of IP-10 mRNA expression by qRT-PCR. qRT-PCR data were normalized relative to GAPDH mRNA expression levels in each respective sample and further normalized to the results from the un-treated group, which was set as 1. Results shown are mean plus SD of three independent experiments.</p

    Kinetic expression of chemokines in aorta after balloon injury.

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    <p>Total RNA was extracted from aorta and used for real time quantitative PCR (qRT-PCR) measurement of CCL5 (A), IP-1 (B) and MCP-1 (C) mRNA expression as well as CCR1 (D), CCR3 (E) and CCR5 (F) mRNA expression. qRT-PCR data were normalized relative to GAPDH mRNA expression levels in each respective sample and further normalized to the results from the un-treated aorta, which was set as 1. Results shown are mean plus SD of three to four independent experiments.</p

    The role of p38 MAPK in CCL5 expression.

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    <p>(A) 1×10<sup>6</sup> A7r5 cells were treated with different amounts of a p38 MAPK antagonist SB203580 for 1 h prior to addition of various stimuli as indicated. An identical amount of dissolvent DMSO was used for negative controls. 6 hrs later, total RNA was extracted for measurement of CCL5 mRNA expression by qRT-PCR. 5 µg of different expression plasmids encoding different molecules involved in the p38 MAPK signaling pathway including p38α (B), and DN-MK2 (C), as well as MLK3, MKK3, and MKK6 (D) or empty vector were transiently transfected with Lipofectamine2000. 48 h after transfection, the cells were treated with LPS (1 µg/ml) plus IFN-γ (10 ng/ml) for 6 h, followed by measurement of CCL5 mRNA expression by qRT-PCR. qRT-PCR data were normalized relative to GAPDH mRNA expression levels in each respective sample and further normalized to the results from the un-treated group, which was set as 1. Results shown are mean plus SD of three independent experiments.</p

    TRIM59 Promotes the Proliferation and Migration of Non-Small Cell Lung Cancer Cells by Upregulating Cell Cycle Related Proteins

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    <div><p>TRIM protein family is an evolutionarily conserved gene family implicated in a number of critical processes including inflammation, immunity, antiviral and cancer. In an effort to profile the expression patterns of TRIM superfamily in several non-small cell lung cancer (NSCLC) cell lines, we found that the expression of 10 TRIM genes including TRIM3, TRIM7, TRIM14, TRIM16, TRIM21, TRIM22, TRIM29, TRIM59, TRIM66 and TRIM70 was significantly upregulated in NSCLC cell lines compared with the normal human bronchial epithelial (HBE) cell line, whereas the expression of 7 other TRIM genes including TRIM4, TRIM9, TRIM36, TRIM46, TRIM54, TRIM67 and TRIM76 was significantly down-regulated in NSCLC cell lines compared with that in HBE cells. As TRIM59 has been reported to act as a proto-oncogene that affects both Ras and RB signal pathways in prostate cancer models, we here focused on the role of TRIM59 in the regulation of NSCLC cell proliferation and migration. We reported that TRIM59 protein was significantly increased in various NSCLC cell lines. SiRNA-induced knocking down of TRIM59 significantly inhibited the proliferation and migration of NSCLC cell lines by arresting cell cycle in G2 phase. Moreover, TRIM59 knocking down affected the expression of a number of cell cycle proteins including CDC25C and CDK1. Finally, we knocked down TRIM59 and found that p53 protein expression levels did not upregulate, so we proposed that TRIM59 may promote NSCLC cell growth through other pathways but not the p53 signaling pathway.</p></div

    IRF-1 specifically binds to the CCL5 promoter.

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    <p>(A) Nuclear extracts were isolated from A7r5 cells following IFN-γ or LPS stimulation for 4 h. EMSA was performed with 10 µg of nuclear extract for each sample and a double-stranded oligonucleotide probe containing the −161/−134 region of the CCL5 promoter (sequence given with the critical IRF-RE underlined). (B) “Supershift” EMSA was performed with the −161/−134 probe and nuclear extracts from IFNγ/LPS-stimulated macrophages with an anti IRF-1 antibody and its control rabbit IgG being used. The IRF-1-related complex is indicated by an arrow.</p

    IRF-1 mediates CCL5 transcription in smooth muscle cells.

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    <p>(A) 5 µg of CCL5 promoter driven luciferase construct was transfected into A7r5 cells by Lipofectamin2000. The transfected cells were treated with different amounts of IFN-γ as indicated, LPS (1 µg/ml), or IFN-γ plus LPS for 7 hours, followed by cell lysis and detection of luciferase activity. (B) 5 µg of WT and IRF-1 mutant CCL5 promoter constructs were respectively transfected into A7r5 cells by Lipofectamine2000 and treated with IFN-γ (10 ng/ml) and LPS (1 µg/ml) for 7 hrs, followed by measurement of luciferase activity in cell lysates. Luciferase activities were normalized to the activities obtained in WT CCL5 promoter transfected cells without treatment which was set as 1. (C) 5 µg of WT and IRF-1 mutant CCL5 promoter constructs were respectively co-transfected with PACT-c (empty vector) and IRF-1 expression plasmid into A7r5 cells by Lipofectamine2000, followed by IFN-γ and LPS treatment. Luciferase activity was measured in cell lysates. All results shown represent mean plus SD of three to four separate experiments.</p
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