*2.3. Cell Viability Assay*

For cytotoxicity testing, 9000 HepaRG cells per well were seeded in the inner 60 wells of a 96-well plate. After proliferation and differentiation, FBS was set to 2% for 48 h before incubation. DMSO concentration of 1.7% was kept to reach the maximum expression of various CYP enzymes and transporters to ensure a strong metabolic activation of PAs [42]. The cells were treated with PAs in different concentrations as indicated in the figures. After 24 h of incubation, 10 μL of undiluted MTT reagent (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide, Sigma-Aldrich, Taufkirchen, Germany) were added per well. After an incubation time of 30 min at 37 ◦C, the supernatant was removed and the formazan crystals were dissolved in 130 μL isopropanol with 0.7% sodium dodecyl sulfate (SDS, Merck, Darmstadt, Germany) for approximately 30 min under shaking and light protection. Absorption was measured at 570 nm, with 630 nm as reference wavelength [43]. Cell viability was calculated by subtracting the background and normalizing the treatment to solvent control. Solvent control was set to 100%. Four independent experiments with three replicates each were performed.

#### *2.4. Isolation of Total RNA and Quantitative Real-Time Polymerase Chain Reaction (qPCR)*

The effect of PAs on the expression of cholestasis-associated genes was analyzed using real-time qPCR. HepaRG cells were seeded at a density of 0.2 × <sup>10</sup><sup>6</sup> cells per well in 6-well plates. After proliferation and differentiation, DMSO and FBS were set to 0.5% and 2% for 48 h before incubation to ensure inducibility of gene expression. The cells were treated with PAs in different concentrations as indicated in the figures. After 24 h of incubation, the cells were washed two times with ice-cold phosphate-buffered saline (PBS). The total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol, including the on-column DNase digestion step. Concentration and purity of the RNA was measured at 260 and 280 nm by a TecanM200Pro using a NanoQuant plate.

cDNA was synthesized by transcribing 1 μg of RNA, using the High Capacity cDNA Reverse Transcriptase Kit according the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). qPCR was conducted with Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), 300 nM primers and 1 μL cDNA per sample in a total volume of 10 μL. The amplification protocol comprised the following steps:


All qPCRs were performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the 384-well format. Primer sequences used for the amplification are summarized in Table S2 in the supplementary material. The primers were obtained from Eurofins (Hamburg, Germany). The results were evaluated according to the

2–ΔΔCt method [44], normalized to the housekeeping gene GUSB (β-glucuronidase) and referred to the solvent control. Three replicates per sample were measured.
