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. 2002 23.
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 pnitroanilide 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%-BSAPBS/0.05%-Tween) for 1 h at room temperature in the dark. Absorption of HRP-substrate (ophenylene-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 of
the 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 Cruz Biotech, 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.