**2. Materials and Methods**

#### *2.1. Chemicals*

Heliotrine was purchased from Latoxan SAS (Valence, France). Echimidine, senecionine, and senkirkine were purchased from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany). All other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany) in the highest purity available.

#### *2.2. Plasmids*

The plasmid pGL4.14-CYP7A1-Prom was constructed as already described [30]. Briefly, the CYP7A1 promoter region was cloned into the vector pGL4.14 (Promega, Madison, WI, USA) upstream of the firefly luciferase reporter gene by means of sequence and ligationindependent cloning [31,32]. The CYP7A1 promoter (−2014 to −1 bp from translation start site) was amplified from human genomic DNA using the primers 5- -CGG TAC CTG AGC TCG CTA GCC AGG AAA GAA CTG CAC CCA TAA T-3 and 5- -CAG ATC TTG ATA TCC TCG AGT TTG CAA ATC TAG GCC AAA ATC T-3 and subsequently inserted between the NheI/XhoI site of pGL4.14. The construction of the Renilla luciferase expression plasmid pcDNA3-Rluc was described elsewhere [33].

### *2.3. Cell Culture*

HepaRG cells were obtained from Biopredic International (Saint-Gregoire, France) and cultured in William's E medium supplemented with 10% (*v/v*) fetal bovine serum (FBS) (both from Pan-Biotech GmbH, Aidenbach, Germany), 100 U/mL of penicillin, 100 μg/mL of streptomycin (Capricorn Scientific GmbH, Ebsdorfergrund, Germany), 5 μg/mL of insulin (Pan-Biotech GmbH, Aidenbach, Germany), and 5 × <sup>10</sup>−<sup>5</sup> M of hydrocortisone hemisuccinate (Sigma-Aldrich, Taufkirchen, Germany) at 37 ◦C in a humidified atmosphere containing 5% CO2. For all experiments, HepaRG cells were used in passages between 17 and 20. After seeding, HepaRG cells were cultivated in the medium for two weeks. To initiate differentiation of the cells, the HepaRG cells were then cultivated with a medium containing 1% dimethyl sulfoxide (DMSO) for two days, followed by a medium with 1.7% DMSO (Merck KGaA, Darmstadt, Germany) for a further 12 days. For investigating PA-induced hepatotoxic effects in HepaRG cells, two different incubation scenarios were performed: a single incubation for 24 h and a repeated incubation for 14 days comprising a total of seven incubations every two days. The exact cultivation and incubation schemes have recently been published [29].

The human hepatocarcinoma cell line HepG2 was obtained from the European Collection of Cell Cultures (ECACC, Porton Down, UK). The cells were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM, PAN-Biotech GmbH, Aidenbach, Germany) supplemented with 10% (*v/v*) FBS (Capricorn Scientific GmbH, Ebsdorfergrund, Germany), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (both from Capricorn Scientific GmbH, Ebsdorfergrund, Germany) at 37 ◦C in a humidified atmosphere containing 5% CO2. At a confluence of about 80–90%, the cells were passaged and plated at a density of 3 to 5 × <sup>10</sup><sup>4</sup> cells/cm2. Cells at passages up to 12 were used for all experiments.
