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: Clinical and Translational Science

Article Title: Characterization of CYP3A pharmacogenetic variation in American Indian and Alaska Native communities, targeting CYP3A4*1G allele function

doi: 10.1111/cts.12970

Figure Lengend Snippet: Allele frequencies in SCF ( n = 363) and YK ( n = 350) populations compared with other populations globally

Article Snippet: CYP3A4 was detected with primary antibody, CYP3A4 Mouse MaxPab (Abnova, Taipei City, Taiwan), diluted (1:1,000) in blocking buffer (Tris‐buffered saline +0.2% Tween +7% nonfat milk), and secondary antibody, HRP‐conjugated anti‐mouse (Thermo Fisher) diluted (1:100,000) in blocking buffer +1% goat.

Techniques: Variant Assay

Journal: Clinical and Translational Science

Article Title: Characterization of CYP3A pharmacogenetic variation in American Indian and Alaska Native communities, targeting CYP3A4*1G allele function

doi: 10.1111/cts.12970

Figure Lengend Snippet: CYP3A4 protein content in lymphoblastoid cell lines (LCLs). Immunoblot of CYP3A4 and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) in LCLs with CYP3A4*1 / *1 , *1 / *3 , and *3 / *3 diplotypes (panel a). Quantitation of CYP3A4 normalized to GAPDH endogenous control (panel b). One‐way analysis of variance was used to compare CYP3A4 diplotypes

Article Snippet: CYP3A4 was detected with primary antibody, CYP3A4 Mouse MaxPab (Abnova, Taipei City, Taiwan), diluted (1:1,000) in blocking buffer (Tris‐buffered saline +0.2% Tween +7% nonfat milk), and secondary antibody, HRP‐conjugated anti‐mouse (Thermo Fisher) diluted (1:100,000) in blocking buffer +1% goat.

Techniques: Western Blot, Quantitation Assay, Control

Journal: Clinical and Translational Science

Article Title: Characterization of CYP3A pharmacogenetic variation in American Indian and Alaska Native communities, targeting CYP3A4*1G allele function

doi: 10.1111/cts.12970

Figure Lengend Snippet: CYP3A4 protein content and activity in human liver microsomes (HLMs). CYP3A4 protein content (panel a) and CYP3A4 metabolic activity (panel b) by CYP3A4 diplotypes: CYP3A4*1 / *1 ( n = 245), *1 / *1G ( n = 64), and *1G / *1G ( n = 10). All incubations were completed in triplicate. Multivariable linear regression was used to compare protein content and activity between HLMs based on genotype, adjusting for site of sample collection

Article Snippet: CYP3A4 was detected with primary antibody, CYP3A4 Mouse MaxPab (Abnova, Taipei City, Taiwan), diluted (1:1,000) in blocking buffer (Tris‐buffered saline +0.2% Tween +7% nonfat milk), and secondary antibody, HRP‐conjugated anti‐mouse (Thermo Fisher) diluted (1:100,000) in blocking buffer +1% goat.

Techniques: Activity Assay

Journal: Clinical and Translational Science

Article Title: Characterization of CYP3A pharmacogenetic variation in American Indian and Alaska Native communities, targeting CYP3A4*1G allele function

doi: 10.1111/cts.12970

Figure Lengend Snippet: Multiple linear regression analysis of covariates with log CYP3A4 protein content and log CYP3A4 activity in human liver microsomes, adjusting for variables independently associated with log CYP3A4 protein content and activity

Article Snippet: CYP3A4 was detected with primary antibody, CYP3A4 Mouse MaxPab (Abnova, Taipei City, Taiwan), diluted (1:1,000) in blocking buffer (Tris‐buffered saline +0.2% Tween +7% nonfat milk), and secondary antibody, HRP‐conjugated anti‐mouse (Thermo Fisher) diluted (1:100,000) in blocking buffer +1% goat.

Techniques: Activity Assay

Journal: Clinical and Translational Science

Article Title: Characterization of CYP3A pharmacogenetic variation in American Indian and Alaska Native communities, targeting CYP3A4*1G allele function

doi: 10.1111/cts.12970

Figure Lengend Snippet: CYP3A4‐mediated vitamin D hydroxylation. Ratio of serum 4ß,25(OH) 2 D 3 and 25(OH)D 3 in unrelated participants from the Yukon‐Kuskokwim (YK) Delta ( n = 514) by CYP3A4 diplotypes: individuals with no CYP3A4*1G ( n = 457) and individuals who at least one copy of CYP3A4*1G ( n = 57). A t ‐test was used to compare the two groups

Article Snippet: CYP3A4 was detected with primary antibody, CYP3A4 Mouse MaxPab (Abnova, Taipei City, Taiwan), diluted (1:1,000) in blocking buffer (Tris‐buffered saline +0.2% Tween +7% nonfat milk), and secondary antibody, HRP‐conjugated anti‐mouse (Thermo Fisher) diluted (1:100,000) in blocking buffer +1% goat.

Techniques:

Journal: Journal of pharmacological sciences

Article Title: SUMOylation of pregnane X receptor suppresses rifampicin-induced CYP3A4 and P-gp expression and activity in LS174T cells.

doi: 10.1016/j.jphs.2015.11.006

Figure Lengend Snippet: Fig. 2. Effect of SUMOylated PXR on RIF-induced PXR transactivation of CYP3A4 and P-gp promoter in reporter gene transfected cells. The LS174T cells were transfected with pGL3- CYP3A4-XREM-Luc (A) or tk-MDR1-Luc (B) reporter gene construct and different expression vectors as indicated in Materials and methods. Cells were treated with vehicle control (0.1% DMSO) or RIF (10 mM) for 24 h. Luciferase activity was then determined. Data are expressed as fold change over the DMSO treated group transfected with empty vector. P values (**P < 0.01, ***P < 0.001) indicate statistically significant when RIF-treated cells in PXR þ SUMO-transfected group were compared to RIF-treated cells in PXR-transfected group (n ¼ 4).

Article Snippet: Protein samples were then separated on 8%e12% SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, USA) that were probed with primary antibodies including CYP3A4 (SC-53850), Pgp (SC-13131) (Santa Cruz Biotechnology) and GAPDH (14C10) (Cell Signaling Technology).

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

Journal: Journal of pharmacological sciences

Article Title: SUMOylation of pregnane X receptor suppresses rifampicin-induced CYP3A4 and P-gp expression and activity in LS174T cells.

doi: 10.1016/j.jphs.2015.11.006

Figure Lengend Snippet: Fig. 3. Effect of SUMOylated PXR on RIF-induced mRNA and protein expression of CYP3A4 and P-gp. The LS174T cells were transfected with different expression vectors as indicated in Materials and methods. Cells were treated with DMSO (0.1%) or RIF (10 mM) for 24 h or 48 h, and then CYP3A4 and P-gp mRNA (AeB) or protein (CeD) levels were analyzed. Data are expressed as fold change over the DMSO treated group transfected with empty vector. P values (*P < 0.05, **P < 0.01, ***P < 0.001) indicate statistically significant when RIF- treated cells in PXR þ SUMO-transfected group were compared to RIF-treated cells in PXR-transfected group (n ¼ 3).

Article Snippet: Protein samples were then separated on 8%e12% SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, USA) that were probed with primary antibodies including CYP3A4 (SC-53850), Pgp (SC-13131) (Santa Cruz Biotechnology) and GAPDH (14C10) (Cell Signaling Technology).

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Journal of pharmacological sciences

Article Title: SUMOylation of pregnane X receptor suppresses rifampicin-induced CYP3A4 and P-gp expression and activity in LS174T cells.

doi: 10.1016/j.jphs.2015.11.006

Figure Lengend Snippet: Fig. 4. Effect of SUMOylated PXR on RIF-induced CYP3A4 enzymatic activity and P-gp transporter activity. LS174T cells were transfected with different expression vectors as indicated in Materials and methods. Cells were treated with DMSO (0.1%) or RIF (10 mM) for 48 h, and then CYP3A4 and P-gp activity was analyzed. (A) Representative and comparative SRM chromatograms of 10-OHMDZ metabolized by CYP3A4 in LS174T cells. (B and C) Data are expressed as fold change over the DMSO treated group transfected with empty vector. P values (*P < 0.05) indicate statistically significant when RIF-treated cells in PXR þ SUMO-transfected group were compared to RIF-treated cells in PXR-transfected group (n ¼ 3).

Article Snippet: Protein samples were then separated on 8%e12% SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, USA) that were probed with primary antibodies including CYP3A4 (SC-53850), Pgp (SC-13131) (Santa Cruz Biotechnology) and GAPDH (14C10) (Cell Signaling Technology).

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation

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

Journal: Gels

Article Title: An Engineered Protein-Based Building Block (Albumin Methacryloyl) for Fabrication of a 3D In Vitro Cryogel Model

doi: 10.3390/gels8070404

Figure Lengend Snippet: Evaluation of liver-specific functions and cellular infiltration of HepG2 cell constructs in cross-sections of 3D BSAMA cryogels at day 1, day 7, and day 14. ( A – C ) Confocal microscopic images of CYP3A4 immunostaining. Blue: nucleus; red: F-actin; green: CYP3A4. ( D – F ) Confocal microscopic images of albumin immunostaining. Blue: nucleus; red: F-actin; green: albumin. Scale bar: 100 μm. ( G ) Distance of the cell migration. ( n = 3, **: p < 0.01, ***: p < 0.005, ****: p < 0.001), compared to the same cryogel type at day 1.

Article Snippet: The samples were incubated with either rabbit anti-albumin primary antibody (1:100, Affinity Biosciences) or mouse anti-CYP3A4 primary antibody (1:100, Affinity Biosciences) in a BSA solution (1% w / v ) at 4 °C overnight.

Techniques: Construct, Immunostaining, Migration

Journal: Gels

Article Title: An Engineered Protein-Based Building Block (Albumin Methacryloyl) for Fabrication of a 3D In Vitro Cryogel Model

doi: 10.3390/gels8070404

Figure Lengend Snippet: Evaluation of liver-specific functions of HepG2 cell constructs in 3D BSAMA cryogels and on 2D substrates at day 1, day 7, and day 14. ( A – C ) Confocal microscopic images of CYP3A4 immunostaining. Blue: nucleus; red: F-actin; green: CYP3A4. ( D – F ) Confocal microscopic images of albumin immunostaining. Blue: nucleus; red: F-actin; green: albumin. Cell constructs in B5-CF displayed the highest protein expression of both CYP3A4 and albumin. Scale bar: 100 μm.

Article Snippet: The samples were incubated with either rabbit anti-albumin primary antibody (1:100, Affinity Biosciences) or mouse anti-CYP3A4 primary antibody (1:100, Affinity Biosciences) in a BSA solution (1% w / v ) at 4 °C overnight.

Techniques: Construct, Immunostaining, Expressing

Journal: Gels

Article Title: An Engineered Protein-Based Building Block (Albumin Methacryloyl) for Fabrication of a 3D In Vitro Cryogel Model

doi: 10.3390/gels8070404

Figure Lengend Snippet: Effect of HepG2 cell culture in 3D BSAMA cryogels and on 2D substrates on liver-specific gene expression at day 14. HepG2 cells were cultured in 3D and 2D culture systems, and their RNA was extracted for the quantitative real-time PCR analysis of AAT, CYP3A7, G6Pase, HNF4α, HNF6, E-cadherin, N-cadherin, ZO-1, claudin-1, CYP3A4, albumin. The data were normalized to the housekeeping gene GAPDH. ( n = 3, *: p < 0.05, **: p < 0.01, ***: p < 0.005, ****: p < 0.001, compared to 2D culture. #: p < 0.05, ##: p < 0.01, ###: p < 0.005, ####: p < 0.001, between the samples at the extremities of the line.) AAT: alpha 1-antitrypsin, G6Pase: glucose 6-phosphatase, HNF: hepatocyte nuclear factors, ZO-1: zonula occludens.

Article Snippet: The samples were incubated with either rabbit anti-albumin primary antibody (1:100, Affinity Biosciences) or mouse anti-CYP3A4 primary antibody (1:100, Affinity Biosciences) in a BSA solution (1% w / v ) at 4 °C overnight.

Techniques: Cell Culture, Gene Expression, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Gene Expression Reprogramming by Citrate Supplementation Reduces HepG2 Cell Migration and Invasion

doi: 10.3390/ijms25126509

Figure Lengend Snippet: RNA-Seq versus RT-qPCR gene expression correspondence.

Article Snippet: The primary antibody specific to the CYP3A5 (13737-1-AP) protein was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Gene Expression Reprogramming by Citrate Supplementation Reduces HepG2 Cell Migration and Invasion

doi: 10.3390/ijms25126509

Figure Lengend Snippet: Migration and Matrigel invasion assay. ( A ) Representative Western blotting and densitometric analysis of CYP3A5 protein upon citrate treatment. β-actin was used as a loading control. Data, expressed as the mean ± SE of four independent experiments, are shown as a fold change compared to control cells. ( B ) Effect of citrate treatment on the invasive and migratory ability of HepG2 cells by Transwell assay (scale bar, 100 μm). Migrating and invaded cells are represented as purple cells dyed with crystal violet. Data are presented as the mean ± SE of three independent experiments. The statistical analysis was performed using the GraphPad Prism 8.4.2 software (unpaired, two-tailed t -test, ** p < 0.01, *** p < 0.001).

Article Snippet: The primary antibody specific to the CYP3A5 (13737-1-AP) protein was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Migration, Invasion Assay, Western Blot, Control, Transwell Assay, Software, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Gene Expression Reprogramming by Citrate Supplementation Reduces HepG2 Cell Migration and Invasion

doi: 10.3390/ijms25126509

Figure Lengend Snippet: Primer sequences used for RT-PCR analysis.

Article Snippet: The primary antibody specific to the CYP3A5 (13737-1-AP) protein was purchased from Proteintech (Rosemont, IL, USA).

Techniques: