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: 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: 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: 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: Oncotarget

Article Title: The associations of genetic polymorphisms in CYP1A2 and CYP3A4 with clinical outcomes of breast cancer patients in northern China

doi: 10.18632/oncotarget.16359

Figure Lengend Snippet: Association of CYP1A2 and CYP3A4 genetic polymorphisms with protein expression

Article Snippet: The sections were incubated overnight at 4°C with primary antibody CYP1A2 (1:100 dilution, a recombinant rabbit monoclonal antibody, BOSTER: PB0574) and CYP3A4 (1:50 dilution, a recombinant rabbit monoclonal antibody, BOSTER: PB1111).

Techniques: Expressing

Journal: Oncotarget

Article Title: The associations of genetic polymorphisms in CYP1A2 and CYP3A4 with clinical outcomes of breast cancer patients in northern China

doi: 10.18632/oncotarget.16359

Figure Lengend Snippet: Prognostic factors in the cox proportional hazards model

Article Snippet: The sections were incubated overnight at 4°C with primary antibody CYP1A2 (1:100 dilution, a recombinant rabbit monoclonal antibody, BOSTER: PB0574) and CYP3A4 (1:50 dilution, a recombinant rabbit monoclonal antibody, BOSTER: PB1111).

Techniques:

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: Two antibodies against CYP3A4 were used: (1) rabbit polyclonal anti-human CYP3A4 (AB1254) (1:1,000; Millipore); (2) rabbit polyclonal anti-human CYP3A4 (CR 3340, 1:1,000, BIOMOL; Enzo Life sciences, U.S.A.).

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: Two antibodies against CYP3A4 were used: (1) rabbit polyclonal anti-human CYP3A4 (AB1254) (1:1,000; Millipore); (2) rabbit polyclonal anti-human CYP3A4 (CR 3340, 1:1,000, BIOMOL; Enzo Life sciences, U.S.A.).

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: Two antibodies against CYP3A4 were used: (1) rabbit polyclonal anti-human CYP3A4 (AB1254) (1:1,000; Millipore); (2) rabbit polyclonal anti-human CYP3A4 (CR 3340, 1:1,000, BIOMOL; Enzo Life sciences, U.S.A.).

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: Two antibodies against CYP3A4 were used: (1) rabbit polyclonal anti-human CYP3A4 (AB1254) (1:1,000; Millipore); (2) rabbit polyclonal anti-human CYP3A4 (CR 3340, 1:1,000, BIOMOL; Enzo Life sciences, U.S.A.).

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: Two antibodies against CYP3A4 were used: (1) rabbit polyclonal anti-human CYP3A4 (AB1254) (1:1,000; Millipore); (2) rabbit polyclonal anti-human CYP3A4 (CR 3340, 1:1,000, BIOMOL; Enzo Life sciences, U.S.A.).

Techniques: Control, Incubation, Expressing

Journal: RSC advances

Article Title: Porcine hepatocytes culture on biofunctionalized 3D inverted colloidal crystal scaffolds as an in vitro model for predicting drug hepatotoxicity.

doi: 10.1039/c9ra03225h

Figure Lengend Snippet: Fig. 4 Evaluation of spatial liver-specific functions of pig hepatocytes. (A and B) Evaluation of pig hepatocytes albumin and urea secretion in 2D and 3D ICC scaffolds at day 2, 8 and 12. The results are mean SD (n ¼ 3). *p < 0.05 and **p < 0.01 compared to 2D culture. (C) Immuno- fluorescence staining of CYP3A4 and albumin in cells on 2D, PEGDA scaffolds, Col I-coated scaffolds and fibronectin-coated scaffolds, on day 14. CYP3A4 was stained with Alexa Fluor 488 (green), albumin was stained with Alexa Fluor 594 (red), and cell nuclei were stained with H33342 (blue). Scale bar ¼ 50 mm. (D) Gene expression of functional liver markers in hepatocytes on ICC scaffolds. Quantitative real-time PCR analysis of the mRNA of specific genes was conducted, and the data were normalized to the housekeeping gene GAPDH. Fold change comparison between the ICC groups and 2D culture; *p < 0.05 and **p < 0.01 compared to 2D culture.

Article Snippet: Samples were successively incubated (4 C, overnight) with either goat anti-albumin primary antibody (1 : 100; Abcam) or rabbit anti-CYP3A4 primary antibody (1 : 200; Merck) in BSA (2% w/v in PBS, 80 mL).

Techniques: Staining, Gene Expression, Functional Assay, Real-time Polymerase Chain Reaction, Comparison