Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Binding of PPARα to the CYP2C8 promoter in vivo . Precipitated DNA from HepaRG cells without (w/o) treatment or after treatment with amodiaquine (10 μM for 6 h) was purified and was used, together with input DNA, as template for Sybr-Green PCR using a total of 12 primer pairs spanning approximately 10 kb of the CYP2C8 promoter region. Raw Ct (cycle threshold) values were normalized to input DNA to calculate the percentage of DNA immunoprecipitated. Primers encompassing the PPRE of the human HMGCR gene were used as positive control. Means relative to negative control primer pair (n.c.) are shown. (A–F) Schematic representation of CYP2C8 promoter regions, which were subjected for the ChIP analysis. Promoter scheme includes binding sites of previously described transcription factors.

Article Snippet: A standard curve was obtained using CYP2C8 cDNA-containing linearized plasmid DNA purchased from OriGene (SC107944).

Techniques: Binding Assay, In Vivo, Purification, SYBR Green Assay, Immunoprecipitation, Positive Control, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Different molecular forms of CYP2C8 in human liver samples. A, panels i–iii, representative immunoblots of mitochondrial and microsomal proteins (50 μg each) from human liver samples (Mt, mitochondrial fraction; Mc, microsomal fraction; HL, human liver used in labeling of some of the samples). Blots were developed with polyclonal antibodies to CYP2C8 (1:500 dilution, v/v) and TOM20 (1:2,000 dilution, v/v) and monoclonal antibody to cytochrome P450 reductase (CPR) (1:1,500 dilution, v/v). In addition to the full-length CYP2C8, a smaller form of 44 kDa (Var_3 (V3)) was seen predominantly in the mitochondrial fraction. The numbers in parentheses below the CYP2C8 immunoblot represent the ratios of Var_3 and full-length (Var_1) proteins in terms of band intensities. B, relative distribution of full-length CYP2C8 in mitochondria and microsomes of the liver samples analyzed in A. The percent distribution was calculated based on the densitometry of band intensities in A. Results represent averages from two blots.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Western Blot, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Identification of the 44-kDa species as a splice variant. A, protein sequence alignment of full-length (WT) CYP2C8 (marked 1) and its two splice variants (marked 2 and 3, respectively). Conserved residues in all three sequences are marked with asterisks. The reported Var_2 and Var_3 lack the N-terminal stretch of amino acids from the full-length protein. B, schematic representation of differential splicing of pre-mRNA for the generation of full-length CYP2C8 (Var_1) and Var_3 mRNAs. Ex, exon; aa, amino acids. C, DNA amplicons generated by RT-PCR of total RNAs using the common 3′- and 5′-primers were resolved on a 2% agarose gel (w/v) and stained with ethidium bromide. WT CYP2C8, Var_3 (V3), and a slow migrating minor component, Var_2 (V2), are shown for the liver samples analyzed in Fig. 1A. M, DNA marker. Relative band intensities of Var_3 and Var_1 amplicons are presented as ratios in parentheses below the gel patterns. C, panels i and ii, immunoblot analysis of mitochondrial proteins (50 μg each) from individual liver samples from Fig. 1A. Mitochondrial protein (Mito) from HepG2 cells expressing Var_3 cDNA was run alongside.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Variant Assay, Sequencing, Generated, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Marker, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: In silico analysis of WT CYP2C8 and CYP2C8 variant *3. A, three-dimensional structures of WT CYP2C8 (WT2C8; in yellow at left), Var_3 (V3-2C8; in pink at center), and Var_3 (V3) superimposed on WT (right). Heme is colored in red, and felodipine is colored blue. B, amino acid residues interacting with the heme (in red) through hydrogen-bonding interactions in WT CYP2C8 (at left) and Var_3 (at right). C, list of amino acid residues (one-letter notation) involved in anchoring the heme in the two molecular forms.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: In Silico, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Different subcellular targeting efficiencies of the three variant proteins. A, in vitro import of 35S-labeled translation products in isolated rat liver mitochondria. Panel i, 35S-labeled translation products of wild type (WT2C8), Var_2 (V2-2C8), and Var_3 (V3-2C8). Radiometric imaging of gels was performed to determine the level of import of input protein for each construct in trypsin-treated samples (T). The input protein level was considered to be 100% in each case. Panel ii, import of dihydrofolate reductase (DHFR) and SU9-dihydrofolate reductase (Su9-DHFR) proteins as negative and positive controls, respectively. The lanes marked “In” or “I” (for input) were loaded with 20% of the counts used for the import reactions. “C” represents control experiments in which total protein bound and imported into mitochondria is present, “T” represents trypsin-treated mitochondria in which only the protein imported into mitochondria is present. B, translocation of WT and variant 2 and 3 proteins in transiently transfected COS-7 cells. Panel i, total cell lysates (50 μg each) from transiently transfected COS-7 cells were resolved by 12% SDS (w/v)-PAGE and probed with antibodies to CYP2C8 and β-actin for assessing loading levels. Panel ii, mitochondria and microsomes were isolated from transfected COS-7 cells, and 50 μg of protein each was resolved by 12% SDS (w/v)-PAGE and probed with antibodies to CYP2C8, cytochrome P450 reductase (CPR), and cytochrome-c oxidase subunit I. Panel iii, relative resistance of mitochondrion-associated proteins to trypsin treatment (T). In some cases, mitochondria were lysed by treatment with 1% Triton X-100 (v/v) before trypsin treatment (TT). Proteins (50 μg each) were resolved by SDS-PAGE and probed with antibodies to CYP2C8 and TOM20 for immunoblot analysis. C, immunofluorescence microcopy of COS-7 cells transfected with WT (Var_1), Var_2, or Var_3 cDNA. Panel i, a–c, co-localization of CYP2C8 with a mitochondrial marker, cytochrome-c oxidase subunit I (CcOI). Cells were stained with a 1:1,000 dilution (v/v) of primary anti-goat antibody to CYP2C8 (green) (Abcam, Cambridge, MA) and co-stained with a 1:500 dilution (v/v) of cytochrome-c oxidase subunit I (red) (anti-mouse) antibody (Abcam). Panel ii, a–c, co-localization of CYP2C8 with microsomal membrane marker calreticulin. Cells were stained with CYP2C8 (green) as above and co-stained with a 1:500 dilution (v/v) of calreticulin (CRT) (anti-rabbit) antibody (red) (Santa Cruz Biotechnology, Santa Cruz, CA). The cells were subsequently incubated with secondary Alexa Fluor 546-conjugated anti-mouse and then anti-rabbit IgG and Alexa Fluor 488-conjugated anti-goat IgG and imaged through a confocal microscope. Numbers in the bottom panels indicate Pearson coefficients for coincidence calculated using Volocity 5.3 software.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Variant Assay, In Vitro, Labeling, Isolation, Imaging, Construct, Translocation Assay, Transfection, SDS Page, Western Blot, Immunofluorescence, Marker, Staining, Incubation, Microscopy, Software

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: The mitochondrial targeting efficiency of N-terminal signals of the full-length (Var_1; WT), Var_2 (V*2), and Var_3 (V*3) proteins The mitochondrial targeting efficiency of the three proteins was analyzed using the MitoProt II-v1.101 program. aa, amino acids.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Reconstitution of catalytic activity of purified WT CYP2C8. A, reconstitution of paclitaxel 6-hydroxylation activity was done with 0.2 nmol of purified CYP protein with or without 0.5 nmol of cytochrome P450 reductase (CPR), 0.4 nmol of purified Adx, 0.04 nmol of purified AdxR, and 10 μm paclitaxel in a 0.3-ml final volume as described under “Materials and Methods.” Montelukast (Mon) (5 μm) and inhibitory antibody to CYP2C8 (2C8Ab) (10 mg/ml) were used. Control ascites fluid (CAF; 10 mg/ml) was used as a negative control. B, reconstitution of dibenzylfluorescein oxidation was carried out essentially as described above in A. The activities in all cases represent the means ± S.E. (error bars) of three to five separate assays. Purified CYP2C8 was preincubated with inhibitors and control ascites fluid as described under “Materials and Methods.” ♦ in A indicates no detectable activity.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Activity Assay, Purification, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Subcellular distribution of CYP2C8 in stable HepG2 cells. A, immunoblot analysis of mitochondrial (Mt) and microsomal (Mc) fractions isolated from stable HepG2 cells with CYP2C8 antibody (middle panel). The blots were also probed with antibodies to TOM20 (bottom panel) as a mitochondrion-specific marker and NADPH-cytochrome P450 reductase (top panel) as a microsome-specific marker. Std, standard. B, membrane-extrinsic and -intrinsic nature of wild-type CYP2C8 (WT 2C8) and Var_3 (V3 2C8) in the mitochondrial (Mito) and microsomal (Micro) fractions was analyzed by the alkaline Na2CO3 extraction method described under “Materials and Methods.” The soluble (E) and insoluble (P) protein fractions were subjected to SDS-PAGE separation and probed with CYP2C8 antibody (2C8Ab) by immunoblot analysis. C, the relative levels of viral vector DNA (puromycin acetyltransferase gene) were determined by real time PCR using total cell DNA as template and the actin gene as an internal reference. D, spectrophotometric scans of CYP heme in the mitochondrial and microsomal fractions obtained from HepG2 stable cells expressing WT and Var_3 proteins. Fe2+-CO versus Fe2+ spectra were recorded as described under “Materials and Methods.” Abs, absorbance. E, relative CYP contents of mitochondria and microsomes from mock-, wild-type CYP2C8-, and CYP2C8 Var_3-expressing HepG2 cells. CYP content was measured by CO difference spectra as described under “Materials and Methods.” F, paclitaxel 6-hydroyxlation activity reconstituted with mitochondria and microsomes from stable HepG2 cells expressing WT CYP2C8 and Var_3 CYP2C8 and mock-transfected cells. Assays were carried out as described under “Materials and Methods.” G, ROS production in isolated mitochondria from stable HepG2 cell lines with or without treatment with the antioxidant N-acetylcysteine (NAC) or the inhibitors proadifen and montelukast. Mitochondria (50 μg each) were seeded in 96-well plates for ROS measurements using the 2′,7′-dichlorodihydrofluorescein (DCF) diacetate method as described under “Materials and Methods.” Results represent means ± S.E. (error bars) of three to four separate assays. * indicates a p value <0.05, and ** represents a p value <0.001. ♦ in F indicates no detectable activity. CPR, cytochrome P450 reductase.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Western Blot, Isolation, Marker, SDS Page, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Oxidation of arachidonic acid by mitochondria and microsomes isolated from stable HepG2 cells expressing Var_1 (WT) CYP2C8 and Var_3 proteins. A–D, the mitochondrial (Mt) and microsomal (MIC) proteins (300–500 μg) were assayed for arachidonic acid metabolism as described under “Materials and Methods” using 70 μm arachidonic acid as substrate. Inhibition studies were performed by preincubating enzymes with 5 μm montelukast at 37 °C for 20 min. Reactions were initiated by the addition of 1 mm NADPH and continued for 5 min at 37 °C in a shaking water bath, and the metabolites were extracted and analyzed as described under “Materials and Methods.” Four major arachidonate products (11,12-EET, 14,15-EET, 8,9-EET, and 20-HETE) were quantified as described under “Materials and Methods.” E, mitochondrial (Mito) proteins from Var_3 (V3)-expressing cells were reconstituted with or without added Adx/AdxR or added montelukast using arachidonic acid as substrate. In one case, purified Var_1 (V1) CYP2C8 was reconstituted with Adx/AdxR as described in Fig. 5. The total EET metabolites were quantified using the LC-MS method to ascertain the dependence of the enzyme on Adx/AdxR. The results represent means ± S.E. (error bars) of three independent assays. * indicates p < 0.05, and ** indicates p < 0.001. CPR, cytochrome P450 reductase.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Isolation, Expressing, Inhibition, Purification, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Respiratory dysfunction and ROS generation in cells treated with arachidonic acid. A, panels i–iii, the effect of arachidonic acid on respiration profile was measured using a Seahorse Bioscience XF24 extracellular flux analyzer. All parameters were analyzed using XF software and are displayed as oxygen consumption rates (pmol of O2/min/100 μg of protein) after normalizing for the protein concentration of each well. Panel i, basal OCR accounts for baseline rates of oxygen consumption. Panel ii, 2,4-dinitrophenol-mediated uncoupling generates maximal OCR. Panel iii, inhibition by oligomycin corresponds to ATP-linked OCR. Mean values ± S.E. (error bars) were calculated based on three separate measurements. B, effects of arachidonic acid (AA) on ROS production in stable HepG2 cells. Cells were grown with or without arachidonic acid for 24 h in 6-well plates, and the culture fluids were used for assaying the levels of H2O2 produced using the Amplex Red method as described under “Materials and Methods.” Results represent means ± S.E. (error bars) of three separate readings. * indicates a p value <0.05, and ** indicates a p value <0.001. ♦ in A (panel iii) indicates no detectable activity. V3, Var_3; 2C8, CYP2C8.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Software, Protein Concentration, Inhibition, Produced, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Spearman correlations and population distribution of hepatic CYP2C8 phenotypes. CYP2C8 mRNA was determined by a specific TaqMan real-time RT-PCR assay, protein was determined by Western blotting and enzyme activity was measured by LC–MSMS analysis of amodiaquine N -desethylation in human liver microsomes ( n = 150). Results are means of duplicate measurements. (A–C) Spearman’s rank correlation coefficients are indicated as r s and statistical significance for all comparisons was p < 0.0001. (D) Histograms showing population distributions for mRNA (top), protein (middle), and enzyme activity (bottom) using 20 bins over the entire phenotype range.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Quantitative RT-PCR, Western Blot, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Influence of non-genetic factors on CYP2C8 phenotype.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Influence of PPARα expression and genotype on the CYP2C8 phenotype. (A) Scatter plots of CYP2C8 phenotypes (amodiaquine N-desethylation, CYP2C8 protein and mRNA expression) vs. PPARα mRNA (top) and protein (bottom) expression. Spearman’s rank correlation coefficient r s and corresponding p -values are given. (B) Box-and-whisker plots of CYP2C8 phenotypes (Amodiaquine N -desethylation, CYP2C8 protein and mRNA expression) for two previously described intronic PPARA variants. Genotypes are indicated as G/G, G/A, and A/A (for rs4253728) and A/A, A/G, and G/G (for rs4823613). P (rec.), unadjusted p -value of Wilcoxon–Mann–Whitney test for recessive genetic model; N, number of individuals per group; numbers for the three genotype groups do not add up to 150 due to missing values. Outliers were included in all calculations.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Expressing, Whisker Assay, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: PPARα knockdown and activation in HepaRG cells. (A) mRNA levels were measured in HepaRG cultures cultured in three independent differentiation batches 72 h after PPARα activation using ligand WY14,643 (left part of the diagram) or siRNA-mediated PPARα knock-down (right part of the diagram) and compared with mRNA levels measured in cells treated with either DMSO (left) or non-targeting siRNA (right) set at 1.0. The graph shows the means from three (mRNA, activity) or two (protein) independent experiments, with error bars indicating standard deviations (mRNA, activity) and dots representing individual data points (protein). ∗ , Statistically significant ( P < 0.05, paired t -test). (B) Representative Western Blot analysis of the corresponding protein levels of CYP2C8 following either ligand-mediated induction (lanes DMSO and WY14,643) or siRNA-mediated knock-down (lanes siPPARa and siCTR) of PPARα.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Knockdown, Activation Assay, Cell Culture, Activity Assay, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Binding of PPARα to the CYP2C8 promoter in vivo . Precipitated DNA from HepaRG cells without (w/o) treatment or after treatment with amodiaquine (10 μM for 6 h) was purified and was used, together with input DNA, as template for Sybr-Green PCR using a total of 12 primer pairs spanning approximately 10 kb of the CYP2C8 promoter region. Raw Ct (cycle threshold) values were normalized to input DNA to calculate the percentage of DNA immunoprecipitated. Primers encompassing the PPRE of the human HMGCR gene were used as positive control. Means relative to negative control primer pair (n.c.) are shown. (A–F) Schematic representation of CYP2C8 promoter regions, which were subjected for the ChIP analysis. Promoter scheme includes binding sites of previously described transcription factors.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Binding Assay, In Vivo, Purification, SYBR Green Assay, Immunoprecipitation, Positive Control, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Direct PPARα-mediated regulation of CYP2C8. (A) Electrophoretic mobility shift assays using in vitro translated proteins was used for assessment the binding of fluorescence-labeled double-stranded oligonucleotide probes corresponding to the indicated regions within CYP2C8 promoter (selection out of 15 tested probes is shown). As a positive control for binding, the known PPARα/RXRα binding site of the rat ACOX1 gene was used . Complexes of PPARα/RXRα heterodimers with the oligonucleotides are marked by an arrow. (B) Luciferase reporter gene constructs containing the sequences of the CYP2C8 promoter, PBR-A (–9500/–8500 bp) and PBR-C (–2500 to –3500 bp) were cotransfected with a PPARα expression plasmid and renilla-luciferase expression vector into HuH7 cells. Cells were treated with either 100 μM WY14,643 or vehicle DMSO for 48 h before measurement of firefly/renilla luciferase activities. Firefly luciferase activities were normalized to renilla luciferase activities to consider changes in the transfection variability and to DMSO control, set as 1. Data are means of three independent experiments, each performed in triplicates in 96-well format; (mut), mutated sites; ∗ , statistically significant ( P < 0.05) compared with DMSO treatment set to 1.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Electrophoretic Mobility Shift Assay, In Vitro, Binding Assay, Fluorescence, Labeling, Selection, Positive Control, Luciferase, Construct, Expressing, Plasmid Preparation, Transfection, Control

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Inhibitory role of β-catenin in the ligand-mediated induction of CYP2C8 by PPARα. (A) mRNA (gray bars) and protein (black bars) expression levels of CYP2C8 were analyzed by quantitative real-time qRT-PCR and Western blotting, respectively, following treatment of HepaRG cells with 100 μM of WY14,643 in the presence of β-catenin-targeting siRNA (siCatenin) or non-targeting control (siCTR). Shown are mean values of three independent experiments ±SD. ∗ , Statistically significant ( P < 0.05) compared with DMSO treatment set to 1; #, statistically significant ( P < 0.05) when siCatenin-treated cells compared to control siCTR-treated cells. (B) Representative western blot analysis of CYP2C8 protein expression following WY14,643-mediated activation of PPARα in the absence (lane siCatenin + WY14,643) or presence (lane siCTR + WY14,643) of β-catenin.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Population variability of hepatic CYP2C8 expression phenotypes ( n = 150).

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Expressing, Activity Assay