Journal: European journal of clinical pharmacology

Article Title: A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

doi: 10.1007/s00228-023-03489-1

Figure Lengend Snippet: Fig. 1 CYP3A4 activity in freshly isolated human hepatocyte cultures treated with Vehicle (0.1% DMSO), third trimester plasma (LH) and predicted liver concentrations of female hormones (HH), Rifampin (Rif, 10 µM) and Ketoconazole (Ket, 10 µM). Fold induction values are expressed relative 6β- hydroxylation of testosterone formation rates compared to vehicle treated hepatocytes in the respective batch of hepatocytes. HU1552, HU1527, HU1593, HU1632 and HU12-010 represent CYP3A4 activity in individual batch of hepatocytes and Mean represents the average of activity in 5 batches of hepatocytes. Treatments were compared using ANOVA followed by Tukeys' multi- ple comparison test; ****p < 0.0001

Article Snippet: Rabbit anti-human primary monoclonal antibodies against CYP3A4 and β-actin and anti-rabbit IgG linked with horseradish peroxidase were obtained from Cell Signaling Technology, Inc. (Danvers, MA).

Techniques: Activity Assay, Isolation, Clinical Proteomics, Comparison

Journal: European journal of clinical pharmacology

Article Title: A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

doi: 10.1007/s00228-023-03489-1

Figure Lengend Snippet: Fig. 5 mRNA levels of CYP3A4 measured by qRT-PCR and normal- ized to mRNA expression of GAPDH. Corrected mRNA expression values are expressed as fold increase over vehicle treatment. CYP3A4 mRNA expression in hepatocytes from four donors (HU 1522, HU1527, HU1593 and HU1632) treated with 0.1% DMSO (Vehicle), third trimester plasma (LH) and predicted liver (HH) concentrations of female hormones and collected after 72 hr treatment (with media change every 24 hours). HU1552, HU1527, HU1593 and HU1632 represent mRNA levels in individual batch of hepatocytes and Mean represents the average of mRNA levels in 4 batches of hepatocytes. Treatments were compared using ANOVA followed by Tukeys' multi- ple comparison test; *p < 0.05

Article Snippet: Rabbit anti-human primary monoclonal antibodies against CYP3A4 and β-actin and anti-rabbit IgG linked with horseradish peroxidase were obtained from Cell Signaling Technology, Inc. (Danvers, MA).

Techniques: Quantitative RT-PCR, Expressing, Clinical Proteomics, Comparison

Journal: European journal of clinical pharmacology

Article Title: A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

doi: 10.1007/s00228-023-03489-1

Figure Lengend Snippet: Fig. 6 Spearman correlation of CYP3A4 protein and mRNA expres- sion in freshly isolated human hepatocytes treated with 0.1% DMSO (Vehicle), third trimester plasma (LH) and predicted liver (HH) con- centrations of female hormones (with media change every 24 hours). CYP3A4 protein levels were determined by Western blot followed by densitometric analysis and CYP3A4 mRNA expression was estimated using qRT-PCR. Symbols and line represent individual treatment and regression line respectively

Article Snippet: Rabbit anti-human primary monoclonal antibodies against CYP3A4 and β-actin and anti-rabbit IgG linked with horseradish peroxidase were obtained from Cell Signaling Technology, Inc. (Danvers, MA).

Techniques: Isolation, Clinical Proteomics, Western Blot, Expressing, Quantitative RT-PCR

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Development of Biomimetic Hepatic Lobule-Like Constructs on Silk-Collagen Composite Scaffolds for Liver Tissue Engineering

doi: 10.3389/fbioe.2022.940634

Figure Lengend Snippet: Biocompatibility of 5% composite SFC scaffolds (w/v). The viability staining and H&E staining of cells in both scaffolds (A) . SEM images (B) showing the cell growth pattern on the surface of both scaffolds. IHC detection (C) of ALB and CYP3A4 expression of C3A cells in both scaffolds. Quantitative analysis of IHC staining (D) and functional gene expression of C3A cells in both scaffolds (E) . (* p < 0.05).

Article Snippet: Subsequently, they were incubated overnight at 4°C using the following primary antibodies: mouse anti-human ALB (dilution, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, United States), rabbit anti-human CYP3A4 (dilution, 1:100; Proteintech, China), rabbit anti-human MRP 2 (dilution, 1:50; Proteintech), and Alexa Fluor ® 647 Mouse anti-Human CD31 (dilution, 1:100; BD Biosciences).

Techniques: Staining, Expressing, Immunohistochemistry, Functional Assay, Gene Expression

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Development of Biomimetic Hepatic Lobule-Like Constructs on Silk-Collagen Composite Scaffolds for Liver Tissue Engineering

doi: 10.3389/fbioe.2022.940634

Figure Lengend Snippet: Functional gene and protein expression in different culture conditions using RT-qPCR (A) and IF staining (B) . Higher functional gene expression was found in the 3D co-cultures than in the 2D cultures and 3D monocultures (A) . iHepLPC-Heps in both groups expressed ALB, CYP3A4, and MRP 2 after 14 days of culture (B) . (* p < 0.05).

Article Snippet: Subsequently, they were incubated overnight at 4°C using the following primary antibodies: mouse anti-human ALB (dilution, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, United States), rabbit anti-human CYP3A4 (dilution, 1:100; Proteintech, China), rabbit anti-human MRP 2 (dilution, 1:50; Proteintech), and Alexa Fluor ® 647 Mouse anti-Human CD31 (dilution, 1:100; BD Biosciences).

Techniques: Functional Assay, Expressing, Quantitative RT-PCR, Staining, Gene Expression

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Development of Biomimetic Hepatic Lobule-Like Constructs on Silk-Collagen Composite Scaffolds for Liver Tissue Engineering

doi: 10.3389/fbioe.2022.940634

Figure Lengend Snippet: Functional evaluation of iHepLPC-Heps on composite SFC scaffolds. Albumin secretion of different hepatic cultures was assayed using ELISA (A) . Urea synthesis of iHepLPC-Heps cultured under different conditions (B) . Induction of CYP3A4 (C1) and CYP1A2 (C2) expression in response to stimulation with omeprazole and rifampicin, assayed using RT-qPCR. DiI-LDL uptake and CDCFDA staining (D) of iHepLPC-Heps were examined in both groups. (* p < 0.05).

Article Snippet: Subsequently, they were incubated overnight at 4°C using the following primary antibodies: mouse anti-human ALB (dilution, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, United States), rabbit anti-human CYP3A4 (dilution, 1:100; Proteintech, China), rabbit anti-human MRP 2 (dilution, 1:50; Proteintech), and Alexa Fluor ® 647 Mouse anti-Human CD31 (dilution, 1:100; BD Biosciences).

Techniques: Functional Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Quantitative RT-PCR, Staining

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Development of Biomimetic Hepatic Lobule-Like Constructs on Silk-Collagen Composite Scaffolds for Liver Tissue Engineering

doi: 10.3389/fbioe.2022.940634

Figure Lengend Snippet: IHC detection of ALB, CYP3A4, and MRP 2 expression in both groups after transplantation. Protein expressions increased gradually over time. Compared with monocultures, the co-culture group showed upregulated expressions at each time point. Scale bar, 50 µm.

Article Snippet: Subsequently, they were incubated overnight at 4°C using the following primary antibodies: mouse anti-human ALB (dilution, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, United States), rabbit anti-human CYP3A4 (dilution, 1:100; Proteintech, China), rabbit anti-human MRP 2 (dilution, 1:50; Proteintech), and Alexa Fluor ® 647 Mouse anti-Human CD31 (dilution, 1:100; BD Biosciences).

Techniques: Expressing, Transplantation Assay, Co-Culture Assay

Journal: Scientific Reports

Article Title: Differential effects on human cytochromes P450 by CRISPR/Cas9-induced genetic knockout of cytochrome P450 reductase and cytochrome b5 in HepaRG cells

doi: 10.1038/s41598-020-79952-1

Figure Lengend Snippet: Genotypes of POR, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 in HepaRG cells.

Article Snippet: A. Meyer, University of Basel, CH, unpublished data); mouse anti-human CYB5 monoclonal antibody (sc-130311, Santa Cruz Biotechnology, Dallas, USA); mouse monoclonal anti-human CYP1A2 antibody (clone 26-7-5, a kind gift of Frank Gonzalez, Bethesda, USA); mouse monoclonal anti-human CYP2B6 (#458326 Gentest Corp., Woburn, USA); polyclonal rabbit anti-human CYP2C8 (#Hu-A004 Puracyp Inc., Carlsbad, USA); polyclonal rabbit anti-human CYP2C9 (RDI-Cyp2C9abr, Research diagnosics inc., Flanders, USA); monoclonal mouse anti-human CYP2D6 (Mab 114 ); and a rabbit polyclonal anti-human CYP3A4 antibody (#458234 Gentest Corp.).

Techniques: Variant Assay, Sequencing, In Vitro, Expressing

Journal: Scientific Reports

Article Title: Differential effects on human cytochromes P450 by CRISPR/Cas9-induced genetic knockout of cytochrome P450 reductase and cytochrome b5 in HepaRG cells

doi: 10.1038/s41598-020-79952-1

Figure Lengend Snippet: CYP activities in microsomal fractions of HepaRG -POR cells. ( a ) HepaRG cells transduced with sgRNA POR#1 (grey) or POR#2 (white) were differentiated for 3 weeks and harvested for microsome preparation. Enzyme activities of seven CYP enzymes were determined simultaneously by cocktail LC–MS/MS assay and given relative to HepaRG VC . Results are shown as means ± SD of four independent experiments. Statistical significance unpaired t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). ( b-e ) Kinetic analysis of selected substrate conversions in HepaRG microsomes. HepaRG cells transduced with vector control (VC, filled square), sgRNAs POR#1 (filled triangle) and POR#2 (open circle) were differentiated for 3 weeks and harvested for microsome preparation; ( b ) amodiaquine (CYP2C8); ( c ) tolbutamide (CYP2C9); ( d ) atorvastatin (CYP3A4); ( e ) midazolam (CYP3A4). Data were analyzed by Michaelis–Menten model ( b – d ) or by substrate inhibition model ( e ).

Article Snippet: A. Meyer, University of Basel, CH, unpublished data); mouse anti-human CYB5 monoclonal antibody (sc-130311, Santa Cruz Biotechnology, Dallas, USA); mouse monoclonal anti-human CYP1A2 antibody (clone 26-7-5, a kind gift of Frank Gonzalez, Bethesda, USA); mouse monoclonal anti-human CYP2B6 (#458326 Gentest Corp., Woburn, USA); polyclonal rabbit anti-human CYP2C8 (#Hu-A004 Puracyp Inc., Carlsbad, USA); polyclonal rabbit anti-human CYP2C9 (RDI-Cyp2C9abr, Research diagnosics inc., Flanders, USA); monoclonal mouse anti-human CYP2D6 (Mab 114 ); and a rabbit polyclonal anti-human CYP3A4 antibody (#458234 Gentest Corp.).

Techniques: Transduction, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Control, Inhibition

Journal: Scientific Reports

Article Title: Differential effects on human cytochromes P450 by CRISPR/Cas9-induced genetic knockout of cytochrome P450 reductase and cytochrome b5 in HepaRG cells

doi: 10.1038/s41598-020-79952-1

Figure Lengend Snippet: Calculated kinetic parameters of selected substrate conversions.

Article Snippet: A. Meyer, University of Basel, CH, unpublished data); mouse anti-human CYB5 monoclonal antibody (sc-130311, Santa Cruz Biotechnology, Dallas, USA); mouse monoclonal anti-human CYP1A2 antibody (clone 26-7-5, a kind gift of Frank Gonzalez, Bethesda, USA); mouse monoclonal anti-human CYP2B6 (#458326 Gentest Corp., Woburn, USA); polyclonal rabbit anti-human CYP2C8 (#Hu-A004 Puracyp Inc., Carlsbad, USA); polyclonal rabbit anti-human CYP2C9 (RDI-Cyp2C9abr, Research diagnosics inc., Flanders, USA); monoclonal mouse anti-human CYP2D6 (Mab 114 ); and a rabbit polyclonal anti-human CYP3A4 antibody (#458234 Gentest Corp.).

Techniques:

Journal: Scientific Reports

Article Title: Differential effects on human cytochromes P450 by CRISPR/Cas9-induced genetic knockout of cytochrome P450 reductase and cytochrome b5 in HepaRG cells

doi: 10.1038/s41598-020-79952-1

Figure Lengend Snippet: In depth analysis of CYP2C8-mediated amodiaquine N-deethylation. ( a ) Inhibition of amodiaquine N-deethylation with montelukast in microsomal protein. HepaRG cells transduced with vector control (VC, filled circle), sgRNAs POR#1 (filled triangle) and POR#2 (open circle) were differentiated for 3 weeks and harvested for microsome preparation. Inhibition parameters IC 50 and K i were determined by one site competition model. ( b – e ) Kinetic analysis of selected substrate conversions in bactosomes containing recombinant CYP enzymes coexpressed with high (filled circle) or low levels filled square) of POR: ( b ) amodiaquine (CYP2C8); ( c ) tolbutamide (CYP2C9); ( d ) atorvastatin (CYP3A4); ( e ) midazolam (CYP3A4). Data were analyzed by Michaelis–Menten model ( b – d ) or by substrate inhibition model ( e ). ( f ) Relative CYP-activities in microsomal preparations of differentiated HepaRG cells transduced with VC (dark grey), sgRNA POR#1 (light grey) and POR#2 (white) with either NADPH (set to 1.0) or NADH as cofactors. Results are means ± SD of 4 independent experiments. Statistical significance was assessed by unpaired t-test (* p < 0.05, ** p < 0.01).

Article Snippet: A. Meyer, University of Basel, CH, unpublished data); mouse anti-human CYB5 monoclonal antibody (sc-130311, Santa Cruz Biotechnology, Dallas, USA); mouse monoclonal anti-human CYP1A2 antibody (clone 26-7-5, a kind gift of Frank Gonzalez, Bethesda, USA); mouse monoclonal anti-human CYP2B6 (#458326 Gentest Corp., Woburn, USA); polyclonal rabbit anti-human CYP2C8 (#Hu-A004 Puracyp Inc., Carlsbad, USA); polyclonal rabbit anti-human CYP2C9 (RDI-Cyp2C9abr, Research diagnosics inc., Flanders, USA); monoclonal mouse anti-human CYP2D6 (Mab 114 ); and a rabbit polyclonal anti-human CYP3A4 antibody (#458234 Gentest Corp.).

Techniques: Inhibition, Transduction, Plasmid Preparation, Control, Recombinant

Journal: Scientific Reports

Article Title: Differential effects on human cytochromes P450 by CRISPR/Cas9-induced genetic knockout of cytochrome P450 reductase and cytochrome b5 in HepaRG cells

doi: 10.1038/s41598-020-79952-1

Figure Lengend Snippet: CYP expression analysis in HepaRG -POR microsomes or cell lysates. ( a ) Exemplary Western Blots of microsomal fractions of HepaRG cells transduced with vector control (VC) or sgRNAs POR#1 or POR#2 after differentiation for 3 weeks (see Supplementary Fig. online for full blots). ( b ) Means ± SD of protein expression data of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4 of 4 independent preparations are shown relative to VC set to 1.0. Statistical significance was assessed by unpaired t-test. ( c ) Gene expression analysis of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 in HepaRG cells transduced with sgRNAs POR#1 and POR#2 and vector control (VC) and differentiated for 2 weeks was performed by qPCR. Data of 6 independent experiments were normalized to the geometric mean of GAPDH, RPLP0 and β-actin. Statistical significance was assessed by paired t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: A. Meyer, University of Basel, CH, unpublished data); mouse anti-human CYB5 monoclonal antibody (sc-130311, Santa Cruz Biotechnology, Dallas, USA); mouse monoclonal anti-human CYP1A2 antibody (clone 26-7-5, a kind gift of Frank Gonzalez, Bethesda, USA); mouse monoclonal anti-human CYP2B6 (#458326 Gentest Corp., Woburn, USA); polyclonal rabbit anti-human CYP2C8 (#Hu-A004 Puracyp Inc., Carlsbad, USA); polyclonal rabbit anti-human CYP2C9 (RDI-Cyp2C9abr, Research diagnosics inc., Flanders, USA); monoclonal mouse anti-human CYP2D6 (Mab 114 ); and a rabbit polyclonal anti-human CYP3A4 antibody (#458234 Gentest Corp.).

Techniques: Expressing, Western Blot, Transduction, Plasmid Preparation, Control, Gene Expression