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Article Snippet: A. Meyer, University of Basel, CH, unpublished data); mouse anti-human CYB5 monoclonal antibody (sc-130311, Santa Cruz Biotechnology, Dallas, USA); mouse monoclonal anti-human CYP1A2 antibody (clone 26-7-5,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Article Snippet: A. Meyer, University of Basel, CH, unpublished data); mouse anti-human CYB5 monoclonal antibody (sc-130311, Santa Cruz Biotechnology, Dallas, USA); mouse monoclonal anti-human CYP1A2 antibody (clone 26-7-5,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