Cyp3a4

Immunofluorescence staining was performed after 7 days of drug treatment to investigate tissue integrity and presence of cell-specific markers. Membranes were excised from biochips and cells were fixed with ROTIHistofix 4% (Carl Roth) for 10 min at RT. Permeabilization and blocking was performed by adding PBS (Lonza) containing 0.1% saponin (Carl Roth) and 3% normal donkey serum (Abcam, Cambridge, UK) for 30 min at RT. The membrane was subsequently divided with scissors to independently stain the vascular and hepatic cell layers with the primary antibody solution. Primary antibodies ASGPR1 (BD Biosciences, Heidelberg, Germany), CYP3A4 (Sigma-Aldrich), α-GST (BIOZOL, Eching, Germany), CD32b (BIOZOL) and CD206 (Abcam) were incubated at 4 °C overnight. Membranes were washed with PBS/0.1% saponin and incubated with secondary antibodies DAPI (Thermo Fisher Scientific), donkey-anti-mouse-AF647 (Thermo Fisher Scientific), donkey-anti-rabbit-Cy3 (Jackson ImmunoResearch, Cambridgeshire, UK) and donkey-anti-goat-AF488 (Thermo Fisher Scientific) for 1 h at RT. Stained membranes were washed twice with PBS/0.1% saponin, once with PBS and lastly with AQUA AD iniectabilia (B. Braun, Melsungen, Germany). Samples were embedded in fluorescent mounting medium (Agilent Technologies, Waldbronn, Germany).

(±)-Doxazosin mesylate [(±)-DOX], (+)-doxazosin mesylate [(+)-DOX], and (−)-doxazosin mesylate [(−)-DOX] standards (>99.9% purity) were provided by the New Drug Research and Development Center of the North China Pharmaceutical Group Corporation (Shijiazhuang, China). β-Nicotinamide adenine dinucleotide phosphate hydrate (NADP), glucose-6-phosphate dehydrogenase from Baker’s yeast (S. cerevisiae), glucose-6-phosphate, prazosin (internal standard, IS), phenacetin, tolbutamide, and human CYP enzymes (CYP3A4, CYP2D6, CYP2C19, CYP2C8, CYP1A2, CYP2E1, and CYP2C9) were purchased from Sigma-Aldrich (St Louis, USA). Dextromethorphan hydrobromide, paclitaxel, and testosterone were provided by TCI Shanghai (Shanghai, China). Chlorzoxazone and omeprazole were purchased from J&K Chemical (Beijing, China). Heparin sodium injection was provided by the Jiangsu Wanbang Biochemical Pharmaceutical Group Co., Ltd. (Xuzhou, China). Human and rat liver microsomes were purchased from BD Gentest (Franklin, USA). Tris base and MgCl₂ were purchased from Tianjin Yongda Chemical Reagent Co., Ltd. (Tianjin, China). Ultrapure water was prepared by Thermo Scientific Nanopure Water Purifier (Waltham, USA). HPLC-grade methanol and acetonitrile were purchased from Thermo Fisher Scientific (Ottawa, ON, Canada). All the other chemicals were of analytical grade.

CYP3A4 was purchased from Sigma.