Journal: Biochemical pharmacology

Article Title: DEC1 binding to the proximal promoter of CYP3A4 ascribes to the downregulation of CYP3A4 expression by IL-6 in primary human hepatocytes

doi: 10.1016/j.bcp.2012.06.010

Figure Lengend Snippet: (A) Diagrammatic presentation of a series of deleted mutants or 5 base pairs mutants of CYP3A4 luciferase reporter gene constructs. (B) Repressing effect of DEC1 on the CYP3A4-DP-Luc and CYP3A4-P-Luc. (C) Differential repression of CYP3A4-DP-Luc and deleted mutants or 5 base pairs mutants of CYP3A4-P-Luc. HepG2 cells were cultured in 48-well plates and transfected with CYP3A4-DP-Luc (50 ng), CYP3A4-P-Luc (50 ng), or corresponding mutated CYP3A4-P-Luc and along with 5 ng of Null-Renilla reniformis luciferase plasmid. After 48 h incubation, cells were collected and analyzed for luciferase activity. The data are expressed as fold of induction (the ratio of normalized luciferase activity from DEC1 transfection over vector transfection) (n = 4). All experiments were repeated at least three times, and data were expressed as mean ±SD. * p<0.05, statistically significant decrease by IL-6 treatment.

Article Snippet: The activity of CYP3A4 was determined with a P450-GloTM kit (CYP3A4) (Promega, Madison, WI, USA) [ 27 ] according to the manufacturer's manual.

Techniques: Luciferase, Construct, Cell Culture, Transfection, Plasmid Preparation, Incubation, Activity Assay

Journal: Biochemical pharmacology

Article Title: DEC1 binding to the proximal promoter of CYP3A4 ascribes to the downregulation of CYP3A4 expression by IL-6 in primary human hepatocytes

doi: 10.1016/j.bcp.2012.06.010

Figure Lengend Snippet: Primers used for preparation of mutated constructs of CYP3A4-P-luc reporter gene The underlined letters indicated the restriction sites.

Article Snippet: The activity of CYP3A4 was determined with a P450-GloTM kit (CYP3A4) (Promega, Madison, WI, USA) [ 27 ] according to the manufacturer's manual.

Techniques: Construct, Sequencing

Journal: Biochemical pharmacology

Article Title: DEC1 binding to the proximal promoter of CYP3A4 ascribes to the downregulation of CYP3A4 expression by IL-6 in primary human hepatocytes

doi: 10.1016/j.bcp.2012.06.010

Figure Lengend Snippet: Primers used for preparation of mutated constructs of CYP3A4-P92-luc reporter gene The underlined letters indicated the restriction sites and highlight letters indicated mutant sites.

Article Snippet: The activity of CYP3A4 was determined with a P450-GloTM kit (CYP3A4) (Promega, Madison, WI, USA) [ 27 ] according to the manufacturer's manual.

Techniques: Construct, Mutagenesis, Sequencing

Journal: Biochemical pharmacology

Article Title: DEC1 binding to the proximal promoter of CYP3A4 ascribes to the downregulation of CYP3A4 expression by IL-6 in primary human hepatocytes

doi: 10.1016/j.bcp.2012.06.010

Figure Lengend Snippet: (A) Time course of DEC1 mRNA level in human hepatocytes treated with IL-6. Human primary hepatocytes were treated with IL-6 (10 ng/ml) or the same volume of PBS for 24 h. Total RNA was isolated and subjected to qRT-PCR analysis for the level of DEC1 probe as described under Materials and Methods. The qPCR Cts were 25 for DEC1, and 20 for GAPDH. (B) Time course of DEC1 and CYP3A4 protein levels in human hepatocytes treated with IL-6. Human hepatocytes were treated with IL-6 (10 ng/ml) or the same volume of PBS for 48 h, and cell lysates were prepared and lysates (8 μg) were subjected to Western blot analyses with an antibody against DEC1, CYP3A4 or GAPDH (n = 5). All experiments were repeated at least three times, and data were expressed as mean ±SD. * p<0.05, statistically significant decrease by IL-6 treatment.

Article Snippet: The activity of CYP3A4 was determined with a P450-GloTM kit (CYP3A4) (Promega, Madison, WI, USA) [ 27 ] according to the manufacturer's manual.

Techniques: Isolation, Quantitative RT-PCR, Western Blot

Journal: Biochemical pharmacology

Article Title: DEC1 binding to the proximal promoter of CYP3A4 ascribes to the downregulation of CYP3A4 expression by IL-6 in primary human hepatocytes

doi: 10.1016/j.bcp.2012.06.010

Figure Lengend Snippet: (A) (B) Effect of DEC1 knockdown on DEC1 and CYP3A4 mRNA level induced by IL-6. HepG2 cells in 6-well plates were transiently transfected with a mixture containing 800 ng of the DEC1 shRNA construct or the corresponding vector (per well). After 72 h incubation, the transfected cells were treated with IL-6 (10 ng/ml), or PBS for another 24 h. Total RNA was prepared and analyzed for levels of DEC1, CYP3A4 and GAPDH by qRT-PCR. The qPCR Cts were 25 for DEC1, 24 for CYP3A4 and 20 for GAPDH. (C) Effect of DEC knockdown on CYP3A4 protein and catalytic levels induced by IL-6. HepG2 cells were subjected to same procedure as above, cell lysates were analyzed for CYP3A4 (100 μg) by Western blot and its enzymatic activity as described in materials and methods. To determine the DEC1 knockdown efficiency, the cell lysates (80 μg) were analyzed for DEC1 by Western Blot. (D) (E) Effect of DEC1 overexpression on the repression of CYP3A4 and its functional activity. HepG2 cells in 6-well plates were transiently transfected with 800 ng of FlagDEC1 construct or the corresponding vector (per well). After 48 h incubation, the transfected cells, total RNA were isolated and analyzed for the expression of CYP3A4, and GAPDH by qPCR. And cell lysates (100 μg) were analyzed for CYP3A4 expression by Western bolt and its enzymatic activity. To determine the transfection efficiency, the cell lysates (2 μg) were analyzed for DEC1 by Western Blot. All experiments were repeated at least three times, and data were expressed as mean ±SD. * p< 0.05, significant difference from IL-6 treatment and PBS treatment and # p<0.05, significant difference from DEC1 shRNA- or DEC1-transfected cells and vector-transfected cells.

Article Snippet: The activity of CYP3A4 was determined with a P450-GloTM kit (CYP3A4) (Promega, Madison, WI, USA) [ 27 ] according to the manufacturer's manual.

Techniques: Knockdown, Transfection, shRNA, Construct, Plasmid Preparation, Incubation, Quantitative RT-PCR, Western Blot, Activity Assay, Over Expression, Functional Assay, Isolation, Expressing

Journal: Biochemical pharmacology

Article Title: DEC1 binding to the proximal promoter of CYP3A4 ascribes to the downregulation of CYP3A4 expression by IL-6 in primary human hepatocytes

doi: 10.1016/j.bcp.2012.06.010

Figure Lengend Snippet: (A) DEC1 repressed CYP3A4 promoter in a dose-dependent manner. HepG2 cells were cultured in 48-well plates and transfected with CYP3A4-DP-Luc (50 ng), hPXR (50 ng), corresponding concentration of DEC1 ( 1, 5, 10, 20, 50 ng ) and 5 ng of Null-Renilla reniformis luciferase plasmid. Vector plasmid was used to equalize the mount of plasmid DNA for each transfection. After 24 h incubation, the transfected cells were treated with Rif (10 μM) for another 24 h, and then, the cells were collected and analyzed for luciferase activity. (B) Diagrammatic presentation of a series of wild type and various mutated DEC1 constructs. (C) Differential effect of wild type and various mutated DEC1 on CYP3A4 promoter. HepG2 cells were cultured in 48-well plates and transfected with CYP3A4-DP-Luc (50 ng), hPXR (50 ng), wild type DEC1 or corresponding mutated DEC1 (50 ng) along with 5 ng of Null-Renilla reniformis luciferase plasmid for 24 h, and the transfected cells were treated with Rif (10 μM) for another 24 h, and then, the cells were collected and analyzed for luciferase activity. The data were expressed as normalized luciferase activity (based on Null-Renilla reniformis luminescence signal) (n = 3). All experiments were repeated at least three times, and data were expressed as mean ±SD. # p< 0.05, significantly difference from DEC1-transfected cells and vector-transfected cells.

Article Snippet: The activity of CYP3A4 was determined with a P450-GloTM kit (CYP3A4) (Promega, Madison, WI, USA) [ 27 ] according to the manufacturer's manual.

Techniques: Cell Culture, Transfection, Concentration Assay, Luciferase, Plasmid Preparation, Incubation, Activity Assay, Construct

Journal: Biochemical pharmacology

Article Title: DEC1 binding to the proximal promoter of CYP3A4 ascribes to the downregulation of CYP3A4 expression by IL-6 in primary human hepatocytes

doi: 10.1016/j.bcp.2012.06.010

Figure Lengend Snippet: (A) DEC1 binding to region (−59/−35) of CYP3A4 proximal promoter. (B) DEC1 binding to the CCCTGC sequence of CYP3A4 proximal promoter. HepG2 cells were plated in six-well plates and transfected with Flag-DEC1 or Flag CMV2 800 ng/well overnight. Nuclear extracts were prepared with a nuclear extraction kit (Active Motif). Nuclear proteins (10 μg) were incubated with 32P-labelled oligonucleotides (5'-AGCTCCAGCCCTGCCTCCTTCTCTA -3’) in a final volume of 10 μl containing 1 × DNA-binding buffer. For competition experiment, nuclear extracts were incubated with excess unlabelled wild type probe (5×, 50×) or each unlabelled mutated probe (mut1, 2, 3) (50×) and then mixed with the radioactive-belled probe. For supershift assays, an anti-DEC1 antibody was added after the nuclear extracts were incubated with the radioactive-labelled probe. The protein-DNA complexes were resolved on 6% polyacrylamide gel and visualized by Typhoon 8600 Variable Mode Imager. Three independent experiments were performed.

Article Snippet: The activity of CYP3A4 was determined with a P450-GloTM kit (CYP3A4) (Promega, Madison, WI, USA) [ 27 ] according to the manufacturer's manual.

Techniques: Binding Assay, Sequencing, Transfection, Extraction, Incubation