*2.4. Preparation of RNA and Quantitative Real-Time PCR Analysis (qRT-PCR)*

For gene expression analysis, HepaRG cells were seeded in six-well plates at a density of 2 × 105 cells/well. After differentiation, cells were incubated with 5, 35, or 70 <sup>μ</sup>M of PA or the solvent (1.7% DMSO, 0.7% acetonitril (ACN)) for either 24 h or 14 days. To investigate the effects of PAs on CYP7A1 gene expression in HepG2 cells, 7.5 × <sup>10</sup><sup>5</sup> cells/well were seeded in six-well plates and incubated on the next day with the three abovementioned concentrations of PA for 24 h. Following incubation, HepaRG cells were harvested directly into an RLT buffer from the RNeasy Kit (Qiagen, Hilden, Germany) containing 1% β-mercaptoethanol, while HepG2 cells were harvested into phosphate-buffered saline (PBS) and centrifuged for 5 min at 300× *g*. After centrifugation, the supernatant was discarded, and the HepG2 cell pellet was dissolved in an RLT buffer containing 1% β-

mercaptoethanol. The RNA of HepG2 and HepaRG cells was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). a total of 1 μg of RNA was reverse transcribed into single-stranded cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. Quantitative real-time PCR was performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Darmstadt, Germany) using Maxima SYBR Green/ROX qPCR Master Mix (2×) (Thermo Fisher Scientific, Waltham, MA, USA). Thermal cycling conditions have been described elsewhere [16]. Relative quantification of gene expression was calculated according to the 2−ΔΔCt method [34] by normalizing the Ct values of the PA-treated samples to that of the reference gene ACTB (encoding β-actin) and the solvent-treated samples. an upregulation of gene expression was represented by relative expression values > 1, while a downregulation was reflected by values in the range of 0 < x < 1. For allowing a dimension-matched expression for the up- and downregulation, the reciprocals of the values for downregulation were calculated and are therefore shown as values < −1.

#### *2.5. Transcriptional Activation of CYP7A1 by Dual Luciferase Reporter Gene Assay*

For the CYP7A1 reporter gene assay, HepG2 cells were seeded in 96-well plates at a density of 1.8 × 104 cells/well. After 18–24 h, the cells were transiently transfected using the TransIT-LT1 transfection reagent (Mirus Bio, Madison, WI, USA) with 80 ng of the plasmid pGL4.14-CYP7A1-Prom and 1 ng of the Renilla luciferase expression plasmid (pcDNA3-Rluc) used as internal control for normalization. Four to 6 h after transfection, the cells were treated with four different concentrations of the PAs echimidine, heliotrine, senecionine, and senkirkine (5, 35, 70, or 250 μM) or the solvent (2.5% ACN) for 24 h. The known CYP7A1 promotor activity inhibitor phorbol 12-myristate 13-acetate (PMA, 5 μM) was used as a positive control [35]. After 24 h, the cells were lysed by adding 50 μL of lysis buffer to each well (100 mM of potassium phosphate with 0.2% (*v/v*) Triton X-100, pH = 7) and incubated for 15 min on a plate shaker. After centrifugation, luciferase activity was analyzed as previously described [36]. Firefly luciferase values were normalized to Renilla luciferase values and expressed as fold-induction referred to solvent control.

#### *2.6. Staining of Bile Canaliculi to Assay Canalicular Efflux*

The fluorescent dye CDFDA was used to investigate canalicular efflux in HepaRG cells. The membrane-permeable CDFDA is metabolized by intracellular esterases to CDF, a substrate of ABCC2, resulting in fluorescence labeling of bile canaliculi. Therefore, 5.5 × <sup>10</sup><sup>4</sup> HepaRG cells/well were seeded in 24-well plates. After differentiation, the cells were treated with a PA (5 or 35 μM), the solvent (1.7% DMSO, 0.35% ACN), or the positive control (10 mM of acetaminophen (APAP)) for 24 h or 14 days. Cells were washed three times with Hank's balanced salt solution buffer (HBSS; 5.4 mM of KCl, 0.44 mM of KH2PO4, 0.5 mM of MgCl2 × 6 H2O, 0.41 mM of MgSO4 × 7 H2O, 137 mM of NaCl, 4.2 mM of NaHCO3, 0.34 mM of Na2HPO4, 25 mM of D-glucose, and 10 mM of 4-(2 hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH = 7.4)) prior to incubation with 5 μM of CDFDA for 30 min at 37 ◦C. Supernatants were discarded and cells were washed again three times with an HBSS buffer. The distribution of CDF was analyzed using the fluorescence microscope Axio Observer.D1 (objective EC Plan-Neofluar 5x/0.16 Ph 1) at λex/em = 470/525 nm.

#### *2.7. Staining of the Tight Junction Protein Zonula Occludens-1 (ZO-1) Combined with Nuclei Staining*

HepaRG cells were seeded in 24-well plates on gelatin-coated cover slips (Ø 13 mm) at a density of 5.5 × 104 cells/well to determine effects of PAs on tight junction proteins. Subsequent to the treatment with either 5 or 35 μM of PA (echimidine, heliotrine, senecionine, and senkirkine), the solvent (1.7% DMSO, 0.35% ACN), or the positive control APAP (10 mM) for 24 h, cells were washed twice with PBS and fixed by treatment with ice-cold methanol for 20 min. After two washing steps with PBS, nuclei were stained by using SYTOX™ Orange Nucleic Acid Stain (1 μM) (Thermo Fisher Scientific, Braunschweig,

Germany), and cells were washed again twice with PBS. Subsequently, cells were blocked with 10% bovine serum albumin (BSA) in PBS for 1 h at room temperature. The cells were washed twice with PBS before incubation with the primary antibody ZO-1 (1:400 in blocking solution) (Cell Signaling Technology Europe, B.V., Leiden, Netherlands) for 4 h at room temperature. Following primary antibody incubation, the cells were washed twice in PBS and afterwards incubated with the secondary antibody AlexaFlour488-conjugated Goat Anti-Rabbit IgG Alexa Fluor 488 secondary antibody (1:400 in blocking solution; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h in the dark at room temperature. Coverslips were washed twice in PBS and once in H2O and then mounted onto slides using Vecta Shield HardSet Mounting Medium (Vector Laboratories, Burlingame, CA, USA). Imaging of SYTOX Orange (λex/em = 547/570) and ZO-1/Alexa Fluor 488 (λex/em = 488/519) was performed using the confocal laser scanning microscope Leica TCS SP5 (objective HCX PL APO 63x/1.40 OIL PH3 CS).
