Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: CYP3A4 CDS-luciferase activities are reduced by individual or combined shRNAs in CHL cells. (A) CHL cells co-transfected with CYP3A4 CDS-luciferase reporter, pRL-TK and different shRNA expression plasmids showed 20%–30% lower luciferase activities, compared to cells transfected with the pS-NC plasmid. (B) CHL cells transfected with CYP3A4 CDS-luciferase reporter, pRL-TK and different combinations of shRNA expression plasmids showed 40%–50% lower luciferase activities, compared to cells transfected with the pS-NC plasmid. Firefly luciferase activity was normalized to corresponding Renilla luciferase activity, and the control group was set as 100%. * P <0.05; *** P <0.001 compared to the corresponding control ( n =3 in each group, which refers to the number of independent transfection samples in a representative experiment).

Article Snippet: Antibodies of rabbit anti-human CYP3A4 were provided by AVIVA Systems Biology (California, U.S.A.).

Techniques: Luciferase, Transfection, shRNA, Expressing, Plasmid Preparation, Activity Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: Combined shRNAs are effective in targeting CYP3A4 CDS in HEK293 cells. (A) HEK293 cells transfected with CYP3A4 CDS-luciferase reporter and three shRNA expression plasmids showed about 50% lower luciferase activities, compared to cells transfected with the pS-NC plasmid. *** P <0.001 compared to the control ( n =3 in each group, which refers to the number of independent transfection samples in a representative experiment). (B) Fluorescent microscopic pictures of CYP3A4 expression in HEK293 cells at 48 h after transient transfection with CYP3A4 CDS-luciferase reporter plasmid alone (CYP3A4), along with control plasmid (pS-NC) or three shRNA expression plasmids (S1+S2+S3).

Article Snippet: Antibodies of rabbit anti-human CYP3A4 were provided by AVIVA Systems Biology (California, U.S.A.).

Techniques: Transfection, Luciferase, shRNA, Expressing, Plasmid Preparation, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: The mixture of three shRNA plasmids inhibits endogenous CYP3A4 mRNA expression in HepG2 cells. (A) CYP3A4 mRNA expression was elevated 2-fold in HepG2 cells by rifampicin (50 μmol/L for 48 h), as determined by RT-PCR analysis. (B) and (C) The shRNA expression plasmid mixture suppressed CYP3A4 mRNA levels in HepG2 cells. (D) The shRNA expression plasmid mixture did not change CYP3A5 mRNA expression in the HepG2 cells. Relative CYP3A4/5 mRNA expression level was normalized to corresponding GAPDH mRNA level, and the control group was set as 100%. *** P <0.001, compared to the vehicle control. n =3 in each group, which refers to the number of independent transfection samples in a representative experiment.

Article Snippet: Antibodies of rabbit anti-human CYP3A4 were provided by AVIVA Systems Biology (California, U.S.A.).

Techniques: shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Control, Transfection

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: Expression of endogenous CYP3A4 protein is suppressed by combined shRNAs in HepG2 cells. CYP3A4 protein expression (A) in HepG2 cells (treated with rifampicin) was suppressed by about 50% (B) after the transfection with the shRNA expression plasmid mixture, compared to pS-NC control plasmid. CYP3A4 protein expression level was normalized to corresponding GAPDH level in HepG2 cells, and control group was set as 100%. *** P <0.001, compared to the negative control ( n =3 in each group, which refers to the number of independent transfection samples in a representative experiment).

Article Snippet: Antibodies of rabbit anti-human CYP3A4 were provided by AVIVA Systems Biology (California, U.S.A.).

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Control, Negative Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

doi: 10.1016/j.apsb.2014.08.003

Figure Lengend Snippet: CYP3A4 shRNAs alter the chemosensitivity of cells. Transfection of HepG2 cells with CYP3A4 shRNAs reduces the sensitivity to GA (15:1) or GA (17:1), compared to the cells transfected with control plasmid. Cytotoxicity was determined by the MTT assay. The inhibition rate relative to the control (0%) was calculated for each concentration of drug, and the IC 50 value was estimated by fitting the data to a Hill equation .

Article Snippet: Antibodies of rabbit anti-human CYP3A4 were provided by AVIVA Systems Biology (California, U.S.A.).

Techniques: Transfection, Control, Plasmid Preparation, MTT Assay, Inhibition, Concentration Assay

Journal: Scientific Reports

Article Title: Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

doi: 10.1038/srep21316

Figure Lengend Snippet: ( A ) The conversion of testosterone, to 6β-hydroxy testosterone as a measurement of the enzymatic activity of CYP3A4 was determined by HPLC in the presence of the indicated amounts of c-SWCNTs. ( B ) Citrate-coated silver nanoparticles also inhibit the enzymatic activity of CYP3A4. Data are shown as mean values ± S.D. ( C ) Interaction between CYP3A4 protein and c-SWCNTs. Supernatants were taken after incubation of recombinant human CYP3A4 and c-SWCNTs at the indicated concentrations for 60 min and analyzed by SDS-PAGE. The results show that the CYP3A protein is adsorbed by c-SWCNTs (25 µg/mL).

Article Snippet: To assess for direct interaction of the CNTs with CYP3A4, recombinant human CYP3A4 protein and rabbit anti-human CYP3A4 antibody were purchased from Cypex Ltd.

Techniques: Activity Assay, Incubation, Recombinant, SDS Page

Journal: Scientific Reports

Article Title: Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

doi: 10.1038/srep21316

Figure Lengend Snippet: ( A ) The addition of c-SWCNTs or c-SWCNTs with a corona of bovine serum albumin (BSA) to the reaction mixture after completion of the enzymatic reaction demonstrated that c-SWCNTs do not interfere with the assay ( i.e. , no evidence of adsorption of the metabolite, 6β-hydroxy testosterone). ( B ) The effect of bovine serum albumin (BSA) adsorbed onto c-SWCNTs on the enzymatic activity of CYP3A4. ( C ) The protein corona effect is dependent upon the amount of BSA adsorbed onto the c-SWCNTs. BSA was quantified using the BCA assay. Conversion of testosterone, to 6β-hydroxy testosterone was determined by HPLC. Data are shown as mean values ± S.D. ( D ) SDS-PAGE analysis shows that the recombinant human CYP3A4 protein is adsorbed by c-SWCNTs following 60 min incubation and that this interaction is prevented by BSA in a concentration-dependent manner.

Article Snippet: To assess for direct interaction of the CNTs with CYP3A4, recombinant human CYP3A4 protein and rabbit anti-human CYP3A4 antibody were purchased from Cypex Ltd.

Techniques: Adsorption, Activity Assay, BIA-KA, SDS Page, Recombinant, Incubation, Concentration Assay

Journal: Scientific Reports

Article Title: Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

doi: 10.1038/srep21316

Figure Lengend Snippet: ( A ) TGA was performed to confirm the chemical conjugation of PEG onto c-SWCNTs: a higher weight loss is due to the higher MW of the decomposed chain (see Materials and Methods for details). ( B ) The effect of different molecular weight (MW) polyethylene glycol (PEG) chains (750Da, 5 kDa, 10 kDa) on the c-SWCNT-mediated inhibition of enzymatic activity of CYP3A4, as determined by HPLC-based detection of 6β-hydroxy testosterone. The c-SWCNT concentration was 100 ug/mL in all samples. The data are shown as mean values ± S.D. ( C ) Supernatants were taken after incubation of recombinant human CYP3A4 and c-SWCNTs or 5 kDa PEG-c-SWCNTs and analyzed by SDS-PAGE. Three lanes with CYP3A + c-SWCNTs and three lanes with CYP3A + 5 kDa PEG-c-SWCNTs incubated for 0, 30, or 60 min are shown; the differences between these time-points were minimal, however, indicating that the impact of PEGylation on the interaction with CYP3A4 was prompt.

Article Snippet: To assess for direct interaction of the CNTs with CYP3A4, recombinant human CYP3A4 protein and rabbit anti-human CYP3A4 antibody were purchased from Cypex Ltd.

Techniques: Conjugation Assay, Molecular Weight, Inhibition, Activity Assay, Concentration Assay, Incubation, Recombinant, SDS Page

Journal: Scientific Reports

Article Title: Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

doi: 10.1038/srep21316

Figure Lengend Snippet: The last frames of three independent trajectories of Model 1 (side-by-side; consult main text) running for 120 ns ( A ). The key hydrophobic and aromatic residues of CYP3A4 binding to c-SWCNT of three trajectories ( B ). The red van der Waals (vdW) balls represent the 2e channel and the active center is shown by purple sticks.

Article Snippet: To assess for direct interaction of the CNTs with CYP3A4, recombinant human CYP3A4 protein and rabbit anti-human CYP3A4 antibody were purchased from Cypex Ltd.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

doi: 10.1038/srep21316

Figure Lengend Snippet: The heavy atom contact number ( A ), the root mean square deviation (RMSD) of alpha carbon of CYP3A4 ( B ), the van der Waals (vdW) and Coulomb interaction energy ( C ) and hydrogen bond number ( D ) between carboxylated CNT and CYP3A4 as function of time. Local snapshots showing the π-π stacking interaction ( E ), salt bridge interaction ( F ) and hydrogen binding interaction ( G ) by Phe228, Lys35 and Gly31, respectively. Key residues are shown as colored sticks and as transparent pink surfaces.

Article Snippet: To assess for direct interaction of the CNTs with CYP3A4, recombinant human CYP3A4 protein and rabbit anti-human CYP3A4 antibody were purchased from Cypex Ltd.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

doi: 10.1038/srep21316

Figure Lengend Snippet: Atomic force microscopy (AFM) imaging of ( A ) c-SWCNTs alone, ( B ) CYP3A4-containing bactosomes incubated with c-SWCNTs, ( C ) PEG 5 kDa-c-SWCNTs alone, and ( D ) CYP3A4-containing bactosomes incubated with PEG 5 kDa-c-SWCNTs, suggested that the uncoated c-SWCNTs interact with CYP3A4 via side-walls while the PEG 5 kDa coating on the surface of the c-SWCNTs significantly reduces this interaction (and see , ). AFM images were acquired in a non-contact mode with large scale scans of more than 1 μm 2 . At least three random areas per condition were scanned.

Article Snippet: To assess for direct interaction of the CNTs with CYP3A4, recombinant human CYP3A4 protein and rabbit anti-human CYP3A4 antibody were purchased from Cypex Ltd.

Techniques: Microscopy, Imaging, Incubation

Journal:

Article Title: Cellular localization and functional significance of CYP3A4 in the human epileptic brain

doi: 10.1111/j.1528-1167.2010.02956.x

Figure Lengend Snippet: CYP3A4 expression in human temporal lobe epilepsy (TLE) and primary brain cell culture. (A) Examples of a typical brain TLE section and Cresyl violet staining are shown. Dysplastic neurons are indicated by arrowheads, whereas dotted line indicates the boundary between abnormal (double asterisks) and adjacent relative normal region (single asterisk). (B) DAB staining revealed CYP3A4 expression in BBB endothelial cells and neurons. In absence of primary Ab no signal was observed. (C) The majority of neurons (NeuN+) in the malformed regions (double asterisks) were CYP3A4 positive (see Fig. 2 for quantification). CYP3A4 colocalized with MDR1 at the BBB (arrows) and neurons (arrowheads). (D) CYP3A4 and MDR1 protein expression was evaluated in primary brain endothelial cells (EPI-EC) and astrocytes (EPI-Astro, see Table 1). Note that, in the patients examined, EPI-EC had the higher levels of CYP3A4. We confirmed overexpression of MDR1 in the same cells. Intensity of the bands was normalized by β-actin. Results are expressed as mean ± standard error of the mean (SEM) [two-way analysis of variance (ANOVA)]. The asterisk indicates p < 0.05, control HBMEC set as 100% of CYP3A4 or MDR1.

Article Snippet: DAB and immunofluorescence staining Free-floating brain sections were incubated overnight with rabbit polyclonal anti-human CYP3A4 (AB1254) (1:1000; Chemi-Con, Millipore, Bedford, MA, U.S.A.) at 4°C.

Techniques: Expressing, Cell Culture, Staining, Over Expression, Control

Journal:

Article Title: Cellular localization and functional significance of CYP3A4 in the human epileptic brain

doi: 10.1111/j.1528-1167.2010.02956.x

Figure Lengend Snippet: Expression of CYP3A4 in TS and CA brain specimens. CYP3A4 was colocalized with NEUN, GFAP, and MDR1. (A,B) CYP3A4 was expressed at the BBB of tuberous sclerosis (TS) and cavernous angioma (CA) specimens (see Table 1). We also observed different pattern of CYP3A4 expression in parenchymal cells. In particular, TS and CA brains displayed different extents of glial CYP3A4 staining (arrows and arrowheads in B). CYP3A4 and MDR1 colocalized in neurons and BBB. (C) Quantification of NEUN-CYP3A4 positive cells in TLE, TS, and CA. Note that the majority of neurons were positive for CYP3A4. DAPI staining was used to determine the total number of cells in a given section (*p < 0.05).

Article Snippet: DAB and immunofluorescence staining Free-floating brain sections were incubated overnight with rabbit polyclonal anti-human CYP3A4 (AB1254) (1:1000; Chemi-Con, Millipore, Bedford, MA, U.S.A.) at 4°C.

Techniques: Expressing, Staining

Journal:

Article Title: Cellular localization and functional significance of CYP3A4 in the human epileptic brain

doi: 10.1111/j.1528-1167.2010.02956.x

Figure Lengend Snippet: Pattern of CYP3A4 expression in apoptotic and healthy cells. (A–A1) DAPI nuclear condensation (blue) and levels of CYP3A4 expression (green) were quantified (intensity and number of pixels). Two typical examples of healthy (A) and apoptotic (A1) cells are shown. (B) Note the enlarged nuclei (identified by DAPI) indicating diffuse DNA staining in healthy cells expressing CYP3A4 protein (arrowheads). Note the small condensed nuclei (CYP3A4 negative cells, arrows) reflecting apoptosis or irreversible cell damage. (B1) The extent of DAPI nuclear condensation (luminosity/number of pixel) correlates inversely with CYP3A4 expression. (C) Individual values are shown for each data point. No significant overlap between Gaussian distribution curves was observed among apoptotic and healthy neurons. T-test was used, * = p < 0.05.

Article Snippet: DAB and immunofluorescence staining Free-floating brain sections were incubated overnight with rabbit polyclonal anti-human CYP3A4 (AB1254) (1:1000; Chemi-Con, Millipore, Bedford, MA, U.S.A.) at 4°C.

Techniques: Expressing, Staining

Journal:

Article Title: Cellular localization and functional significance of CYP3A4 in the human epileptic brain

doi: 10.1111/j.1528-1167.2010.02956.x

Figure Lengend Snippet: CYP3A4 transfection results in increased carbamazepine metabolism and cell survival. (A) Western blot confirmed CYP3A4 transfection in HEK cells. (A1) HEK-CYP3A4 cells metabolized CBZ to a greater extent compared to nontransfected HEK. Examples of HPLC traces are provided to show the decrease in CBZ level 72 h after treatment. (B) Cell survival was significantly higher in HEK-CYP3A4+ compared to HEK cells. Cells were incubated up to 72 h with a toxic amount of CBZ. Micrographs show the mean cell counting over time. Representative images are shown in B1. Results are expressed as mean ± SEM (t-test). The asterisks indicate p < 0.05.

Article Snippet: DAB and immunofluorescence staining Free-floating brain sections were incubated overnight with rabbit polyclonal anti-human CYP3A4 (AB1254) (1:1000; Chemi-Con, Millipore, Bedford, MA, U.S.A.) at 4°C.

Techniques: Transfection, Western Blot, Incubation, Cell Counting

Journal:

Article Title: Cellular localization and functional significance of CYP3A4 in the human epileptic brain

doi: 10.1111/j.1528-1167.2010.02956.x

Figure Lengend Snippet: Role of EPI-EC and EPI-Glia in CBZ metabolism. (A) EPI-EC, control HBMEC, and EPI-ASTRO were incubated with CBZ up to 72 h. HPLC-UV analysis revealed that EPI-EC metabolized CBZ to a greater extent compared to the other cell types. No significant changes in CBZ levels were measured in the presence of media alone (not shown). (B) The levels of CYP3A4 expression positively correlated with the amount (μg/mL) of CBZ metabolized. Results are expressed as mean ± SEM (t-test). The asterisks indicate p < 0.05.

Article Snippet: DAB and immunofluorescence staining Free-floating brain sections were incubated overnight with rabbit polyclonal anti-human CYP3A4 (AB1254) (1:1000; Chemi-Con, Millipore, Bedford, MA, U.S.A.) at 4°C.

Techniques: Control, Incubation, Expressing

Journal: Molecular pharmaceutics

Article Title: Species Differences in the Pharmacology and Toxicology of PEGylated Helper-Dependent Adenovirus

doi: 10.1021/mp100216h

Figure Lengend Snippet: PEGylation alters the expression and function of hepatic cytochrome P450 3A (CYP3A) in the baboon. Full blood chemistry panels were run on baboons given either unmodified or PEGylated helper-dependent adenovirus expressing beta-galactosidase at a dose of 5 × 1011 or 3 × 1012 vp/kg. Changes in serum aspartate aminotransaminase (AST) (A) and serum lactate dehydrogenase (LDH) (B) profiles were noted. All other parameters measured (see Experimental Section under Biochemical and Hematological Analysis of Blood) fell within normal limits for each primate (data not shown). (C) Immunoblot analysis of hepatic CYP3A in male baboons 96 h after a single systemic dose of 5 × 1011 or 3 × 1012 vp/kg of either unmodified or PEGylated helper-dependent adenovirus expressing beta-galactosidase. Protein levels are reported as arbitrary units of relative density with respect to a known protein standard. (D) In vitro catalytic activity of CYP3A microsomal proteins 96 h after vector administration as measured by the production of the isoform-specific metabolite, 6β-hydroxytestosterone. Data represent values obtained from one primate per experimental condition.

Article Snippet: 47 Detection of CYP3A protein was achieved using a rabbit polyclonal antipeptide against human CYP3A4 antibody (1:4000, A4100, Xenotech) and a corresponding horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology, Denver, MA).

Techniques: Expressing, Western Blot, In Vitro, Activity Assay, Plasmid Preparation

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: FIGURE 3 | 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 MRP2 (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: FIGURE 7 | 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 MRP2 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 MRP2 (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: FIGURE 8 | 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 MRP2 (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: FIGURE 10 | IHC detection of ALB, CYP3A4, and MRP2 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 MRP2 (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: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Effects of BRD7 or BRD9 knockdown or BRD9 inhibition on PXR-mediated induction of CYP3A4 expression. (A) ShP51 cells were transfected with siRNA for BRD7 (siBRD7) or BRD9 (siBRD9). After incubation for 24 hr, the cells were treated with 10 μM rifampicin or 10 μM simvastatin. ShP51 cells (B), HepaRG cells (C), and human primary hepatocytes (D and E) were treated with rifampicin or simvastatin along with iBRD9. PXR, RXRα and GAPDH (A and B) protein, CYP3A4 and GAPDH mRNA (A-D), and CYP3A4 enzyme activity (E) were evaluated by Western blotting, real-time RT□PCR, and P450-Glo assay. Each column represents the mean ± SD (n =3-4). n refers to biological repeats. * P < 0.05, ** P < 0.01, and *** P < 0.001, compared with NT, † P < 0.05, †† P < 0.01, and ††† P < 0.001, compared with siControl or iBRD9 (−). NT: non-treatment. The experiments were repeated two times with similar results.

Article Snippet: Mouse anti-human PXR (sc-48340), RXRα (sc-515929), CYP3A4 (sc-53850), BRD7 (sc-376180), and Ac-lysine (sc-32268) monoclonal antibodies and the goat anti-mouse Cyp3a (sc-30621) polyclonal antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Knockdown, Inhibition, Expressing, Transfection, Incubation, Activity Assay, Western Blot, Glo Assay

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Effects of iBRD9 on PXR binding to chromatin and changes in chromatin structure. (A) ShP51 cells were treated with 20 μM rifampicin along with 20 μM iBRD9 for 24 hr. SSE analysis followed by Western blotting for BRD9 or PXR was performed. The peaks of band intensity are shown in red. (B) Schematic representation of the upstream of CYP3A4 gene. ShP51 cells were treated with 10 μM rifampicin along with 10 μM iBRD9 for 24 hr. Immunoprecipitation with anti-PXR antibody (C) or anti-BRD9 antibody (D) was performed using chromatin from ShP51 cells. Enrichment of the proximal promoter, distal enhancer, and far enhancer of CYP3A4 was evaluated by real-time PCR. (E) Changes in chromatin structure was evaluated by FAIRE followed by real-time PCR. Each column represents the mean ± SD (n = 3). n refers to biological repeats. * P < 0.05, ** P < 0.01, and *** P < 0.001, compared with NT, †† P < 0.01 and ††† P < 0.001, compared with iBRD9 (−). NT: non-treatment. The experiments were repeated two times with similar results.

Article Snippet: Mouse anti-human PXR (sc-48340), RXRα (sc-515929), CYP3A4 (sc-53850), BRD7 (sc-376180), and Ac-lysine (sc-32268) monoclonal antibodies and the goat anti-mouse Cyp3a (sc-30621) polyclonal antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Effects of iBRD9 on the induction of CYP3A in mouse liver. C57BL/6J or hCYP3A-MAC/hPXR male mice (n = 6) were intraperitoneally treated with 50 mg/kg PCN or 10 mg/kg rifampicin for four consecutive days and intraperitoneally treated with 10 mg/kg iBRD9 every other day. Cyp3a11, Cyp3a25, CYP3A4, and Gapdh mRNA (A, D), and Cyp3a, CYP3A4, and KDEL protein (B, E) levels were evaluated by real-time RT□PCR and Western blotting, respectively. (C, F) Triazolam α- and 4-hydroxylase activities were evaluated as marker activity for Cyp3a and CYP3A4. Each column represents the mean ± SD (n = 6). n refers to biological repeats. ** P < 0.01 and *** P < 0.001, compared with vehicle, † P < 0.05 and †† P < 0.01, compared with iBRD9 (−). The experiments were repeated two times with similar results.

Article Snippet: Mouse anti-human PXR (sc-48340), RXRα (sc-515929), CYP3A4 (sc-53850), BRD7 (sc-376180), and Ac-lysine (sc-32268) monoclonal antibodies and the goat anti-mouse Cyp3a (sc-30621) polyclonal antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Marker, Activity 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

Journal: PLoS ONE

Article Title: Involvement of NF-κB in the reversal of CYP3A down-regulation induced by sea buckthorn in BCG-induced rats

doi: 10.1371/journal.pone.0238810

Figure Lengend Snippet: Rats were treated with BCG (125 mg kg -1 , intravenously, once for 2 wks) or BCG + HRP (50, 100, or 200 mg kg -1 d -1 , oral administration for 13d). Liver proteins were extracted to determine NF-κB, iNOS, and CYP3A expression. Equal amounts (30 μg) of protein were subjected to SDS-PAGE followed by western blot analysis using anti-NF-κB, anti-iNOS, and anti-CYP3A antibodies. Results were normalized to LMNA or GAPDH. NF-κB (A), iNOS (B), and CYP3A (C) protein expression levels in rat liver were measured by western blot. NF-κB, iNOS, and CYP3A expression levels were quantified by ImageQuant software (GE Healthcare Life Science, Little Chalfont, UK). Data represent means ± SD of three independent experiments. Asterisks stars above bars indicate differences between groups.

Article Snippet: Kits for cytoplasmic and nuclear protein extraction, bicinchoninic acid (BCA) protein assay (No. AR0146), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), rat IL-1β ELISA (No. BA2913), TNF-α ELISA (No. BA0527), and rabbit polyclonal antibodies against CYP3A (No. A00339), iNOS (No. EK0394), LMNA(Lamin A/C) (Cat. No. BA1227), and GAPDH (No. BA2913) were obtained from Wuhan Boster Biological Engineering Co. Ltd., Wuhan, China.

Techniques: Expressing, SDS Page, Western Blot, Software

Journal: PLoS ONE

Article Title: Involvement of NF-κB in the reversal of CYP3A down-regulation induced by sea buckthorn in BCG-induced rats

doi: 10.1371/journal.pone.0238810

Figure Lengend Snippet: Rats were treated with BCG (125 mg kg-1, intravenously, once for 2 wks) or BCG + HRP (50, 100, or 200 mg•kg -1 •d -1 , oral administration for 13d). The mRNA expression of CYP3A was measured by real-time PCR. Each bar represents the mean ± SD from three independent experiments (n = 10/group). Asterisks stars above bars indicate differences between groups. The significance of the data was determined by one-way analysis of variance (ANOVA) followed by Tukey’s test.

Article Snippet: Kits for cytoplasmic and nuclear protein extraction, bicinchoninic acid (BCA) protein assay (No. AR0146), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), rat IL-1β ELISA (No. BA2913), TNF-α ELISA (No. BA0527), and rabbit polyclonal antibodies against CYP3A (No. A00339), iNOS (No. EK0394), LMNA(Lamin A/C) (Cat. No. BA1227), and GAPDH (No. BA2913) were obtained from Wuhan Boster Biological Engineering Co. Ltd., Wuhan, China.

Techniques: Expressing, Real-time Polymerase Chain Reaction