Supplementary Material: Methods:

FACS Analysis

All fluorochrome conjugated antibodies and isotype controls were obtained from BD Biosciences, Heidelberg, Germany. Propidiumiodide was purchased from Sigma, Steinheim, Germany. After three washes using ice-cold PBS, cells were incubated with PBS on ice and monitored by light microscopy. When cellular morphology changed from flattened to round, cells were gently detached using a police rubber. After centrifugation (250 g, 4 °C, 5 min) cells were counted and adjusted to 5 x 106 cells/mL in staining buffer (PBS, 0.2 % BSA, 0.1 % NaN3). 50 µL of the cell suspension were incubated for 5 min with FcR-blocking reagent (Miltenyi Biotech, Bergisch-Gladbach, Germany) followed by incubation with fluorochrome labelled antibodies or corresponding isotype controls under light exclusion for 30 min at room temperature. Following three washes in staining buffer cells were analysed using an FACS Calibur BD Biosciences, Heidelberg, Germany. Data were saved and analysed using the free software WinMDI.

Enzyme activities

Alanine aminotransferase (ALT)

The activity of ALT was measured by a two phase reaction consisting of the generation of pyruvate and L-glutamate from L-alanine and alpha-ketoglutarate by ALT and the subsequent quantification of pyruvate generation by LDH-dependent beta-NAD+ formation. The protocol was adapted from Schumann et al. 200223.

For high-throughput assays, the measurement of ALT was performed using a colorimetric assay (AL146) from RANDOX Laboratories Crumlin, Co. Antrim, United Kingdom.

Dipeptidyl-Peptidase (DPP) IV

Measurement of DPP IV was performed using the substrate Gly-L-Pro p-nitroanilide (Sigma Aldrich, Germany).Cells were lysed using Triton-X 100/MOPS and lysates were incubated with Gly-L-Pro p-nitroanilide in Hepes-buffered saline. Generation of p-nitroaniline from Gly-L-Pro p-nitroanilide was determined in a multiwall reader at 405 nm absorption.A standard curve with p-nitroaniline was performed to determine activity. Unit Definition: One unit will produce 1.0 µmole of p-nitroaniline from Gly-L-Pro p-nitroanilide per min in 100 mM Tris-HCl at pH 8.0 at 37 °C. Values were normalized for protein content.

Gamma-Glutamyl-Transpeptidase (Gamma-GT)

Gamma-glutamyl-transpeptidase activity was measured using standard protocol24. Cells were cultured in a medium without additives 24 h before the experiment. After rinsing 3 times with ice-cold PBS, cells were lysed with Triton-X 100/MOPS, centrifuged (13.000 U/min, 10 min) and gamma -GT-activity was measured from cell lysate and normalized for protein content.

Lactate-Dehydrogenase (LDH)

LDH-activity was determined spectrophotometrically by measurement of beta-NAD+ from the LDH catalyzed reaction of pyruvate and beta-NADH to lactate and beta -NAD+, using standard protocol (Bergmeyer HU, Bernt E (1974) Methoden der enzymatischen Analyse, 3rd edn. Verlag Chemie. Weinheim, pp 607–612.).

Urea synthesis

Secretion of urea in the cell culture supernatant was determined using QuantiChromTM Urea Assay Kit (BioAssay Systems, Hayward, USA)according to manufacturers instructions. After 24 h incubation in media without additives in presence or absence of different concentrations of ammonium chloride, supernatants were collected and urea was measured according to manufacturer’s instructions.

Toxicity experiments

Toxicity was determined by release of LDH measured using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, USA) according to manufacturers instructions. LDH-release was calculated as percentage of LDH released in the culture media of total LDH (media and lysates).

Cytochrome (CYP) P450 activities and induction

Activities of the CYP P450 enzymes 3A4, 2C9 and 1A2 were determined using P450-GloTM Assay (Promega, Madison, USA) according to manufacturer’s instructions. Briefly, cells were induced for various periods with known CYP-inducers in media without additives25. Luminescence values were normalized for protein content.

Factor VII-ELISA

For analysis of secreted factor VII, a direct ELISA was performed. Cells were incubated with control or different concentrations of menadione for 24 h. First Antibody: Anti-Factor VII (rabbit polyclonal antibody, ABCAM) diluted 1:1000 in 2%-BSA-PBS/0.05% Tween-20 for 2 h at 4°C. Detection antibody: Anti-Rabbit-Peroxidase-Antibody (Santa Cruz, USA) in 1:5000 dilutions (2%-BSA-PBS/0.05%-Tween) for 1 h at room temperature in the dark. Absorption of HRP-substrate (o-phenylene-diamine) was measured at 490 nm, absorption at 630 nm was used as reference wavelength. OD was normalized for protein content.

Western Blotting

For western blot analysis, cells were lysed using RIPA-buffer. After determination of protein content ofthe lysates using the BCA-method, amounts of 20 µg total protein were used per lane. Detection antibody: donkey anti-rabbit-HRP conjugate 1:20.000; ALEXIS / AXXORA GmbH, Lörrach, Germany. Using Pierce ECL Western Blotting Substrate (Thermo Scientific) the signal was transferred to x-ray film.ImageJ Software was used for quantification. First antibodies: Anti-CYP2E1 rat/human, rabbit polyclonal Antibody, Tebu Bio, Germany, 1:1000. Anti-CYP2C9 rat/human, rabbit polyclonal Antibody, ABCAM, UK, 1:1000. Anti-CYP3A4 rat/human, rabbit polyclonal Antibody, Tebu Bio, Germany, 1:1000. Anti-beta-Actin, mouse monoclonal Antibody, Santa CruzBiotech, USA, 1:5000. Secondary Antibodies: goat-Anti mouse IgG, HRP-conjugate, Calbiochem, USA, 1:10.000. Goat-anti-rabbit IgG,HRP-conjugate, Santa Cruz, USA, 1:5.000

Microarray experiments

Microarray experiments were performed using the OHS-401 platform provided by SA Biosciences. Total RNA from primary human hepatocytes on day4 after isolation and MH cells of the same donors on culture day 14 (n=3) was isolated using the Kit provided by SA Biosciences. C-RNA synthesis was performed accoridung to manufacturers instructions and array membranes were hybridized following the instructions of the manufacturer. Signal detection was performed usin X-ray film and after scanning and conversion into TIF-files, data were analysed using TIGR-spotfinder software.

The gene table of OHS-401 is provided in supplements, as well as the raw data of the arrays.

Singal values were normalized to beta-actin signal and expressed as LOG2 of the normalized data.