Hepatocytes were seeded (3.5 × 105 cells/well) on 24-well collagen I-coated plates (BD Biocoat). After 2–3 h, non-attached cells were removed and a top layer of Matrigel™ (250 μg/ml; BD #356237) diluted in serum-free medium (DMEM/F12 supplemented with sodium pyruvate (Gibco), 1X Insulin/Transferrin/Selenium (Gibco), 0.03 μM dexamethasone, Regorafenib 1% Pen/Strep, albumin solution from bovine serum (Sigma)) was applied with pre-cooled pipette. Medium was changed every 24 h. Hepatocyte morphology was monitored daily. Other layers of Matrigel™ were added at day 4 and 8 and 12 of culture. For selected experiments rat hepatocytes were cultured in presence

of DMEM/F12 supplemented with Recombinant Human Epidermal Growth Factor (hEGF, Invitrogen) or 0.5% FCS and with HCM™ Bullet Kit (Hepatocyte Culture Medium, Lonza). The following compounds chosen from a training set used in the 7th EU Framework project Predict-IV Olaparib were used for the long-term term treatment: Cyclosporin A, Metformin (Calbiochem, Switzerland);

Rosiglitazone, Troglitazone (Cayman Chemicals, USA); Amiodarone, Chlorpromazine hydrochloride, Fenofibrate, Ibuprofen, Acetaminophen, Valproic Acid sodium salt (Sigma–Aldrich, Germany). Non-cytotoxic concentrations were chosen (Table 2) and rat hepatocytes were exposed 14 days to perform chronic treatment. The treatments started 24 h after cell seeding. All compounds were dissolved in DMSO and added to the medium with a final concentration of 0.1% vol/vol DMSO. Cells incubated in the presence of 0.1% vol/vol DMSO were used as control. ATP assay: ATP was measured with CellTiter-Glo® Luminescent Cell Viability Assay (Promega, USA) according to manufacturer’s instructions. Lactate Dehydrogenase (LDH) release: LDH

release was measured with Cytotoxicity Detection KitPlus (Roche, Germany) according to manufacturer’s DAPT research buy instructions. Urea synthesis: Urea synthesis was measured with Biochain’s Urea Assay Kit (Biochain, USA) according to manufacturer’s instructions. Albumin secretion: Albumin content was assessed with Rat Albumin ELISA Quantitation Set (Bethyl Laboratories (Montgomery, TX, USA) according to the manufacturer’s instructions. All fluorescence microscopy images of were taken with Thermo Scientific Cellomics™ Arrayscan® VTI, with XF93 Hoechst, FITC, TRITC excitation/emission filters and 10×/20× objective. An amount of at least 2000 cells per well were imaged (8–10 images/well with a 10× objective; 15–20 images/well with a 20× objective). Fluorescence intensities were quantified by using Spot Detector and Compartmental Analysis BioApplication calculating the sum of average fluorescence intensity within a ring surrounding nuclei with a radius of 15 μm for each cell, followed by division of total number of cells measured. Mrp2-mediated transport measurement: Cells were washed twice and incubated with pre-warmed HBSS (+Ca2+/Mg2+) 10 min at 37 °C.