*2.1. Chemicals*

The PAs Lc and senecionine (Sc) (>95% purity) were obtained from PHYTOPLAN Diehm & und Neuberger GmbH (Heidelberg, Germany). In order to get 5 mM stock solutions, the PAs were dissolved in 50% acetonitrile (ACN) and 50% water (*v*/*v*). All other chemicals were purchased from Merck KGaA (Darmstadt, Germany) and Thermo Fisher Scientific (Waltham, MA, USA).

#### *2.2. Cell Culture*

The human hepatoma cell line HepaRG was purchased from Biopredic International (Saint-Grégoire, France). The cells were cultivated at 37 ◦C in a humidified atmosphere of 5% CO2 [39]. After seeding, cells were cultivated in William's Medium E with stable glutamine (PAN-Biotech, Aidenbach, Germany) for proliferation. The medium was supplemented with 10% Fetal Bovine Serum (FBS; PAN-Biotech, Aidenbach, Germany), 50 μM hydrocortisone hemisuccinate (Merck KGaA, Darmstadt, Germany), 100 U/mL penicillin and 100 μg/mL streptomycin (Capricorn Scientific, Ebsdorfergrund, Germany) and 5 μg/mL human insulin (PAN-Biotech, Aidenbach, Germany). After two weeks of proliferation, 1.7% of DiMethyl SulfOxide (DMSO; Merck KGaA, Darmstadt, Germany) was added to initiate differentiation and HepaRG cells were cultivated for another two weeks to fully differentiate before being used for experiments.

#### *2.3. Liver-Specific miRNA Array*

For liver-specific miRNA expression screening, HepaRG cells were seeded at a density of 0.2 × 106 cells per well in 6-well plates and cultivated as described in Section 2.2. After four weeks of cultivation and 48 h prior to incubation, fully differentiated cells were adapted to treatment medium containing 1.7% DMSO and a reduced FBS concentration of 2%. The cells were incubated with 10, 35 and 250 μM of the most potent Lc for 8 h or with 2.5, 10 and 35 μM Lc for 24 h, respectively. The miRNA was isolated using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, including a purification step of the miRNA-enriched fraction using the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany). Initially, cells were lysed with 700 μL QIAzol lysis reagent. As a miRNA isolation efficiency control, a spike-in control consisting of three synthetic templates in different concentrations (Qiagen, Hilden, Germany) was added to the lysis buffer. The subsequent procedure was as follows: incubation for 5 min at Room Temperature (RT), addition of 140 μL chloroform per sample and thoroughly mixing, incubation for 3 min at RT, centrifugation for 15 min at 12,000× *g* at 4 ◦C, transfer of the upper aqueous phase intro a new tube, addition of one volume of 70% ethanol (usually 350 μL) and thorough mixing, transfer of the sample into an RNeasy Mini spin column, and centrifugation for 15 s at 8000× *g* at RT. The RNeasy Mini spin columns containing the mRNA fractions were discarded. The flow-through containing the smaller miRNA fraction was purified as follows: addition of 450 μL 100% ethanol, thorough mixing, transfer of the sample into an RNeasy MinElute spin column and centrifugation for 15 s at 8000× *g* at RT, washing of column with 700 μL RWT buffer and 500 μL RPE buffer, respectively, for 15 s at 8000× *g* at RT, final washing with 500 μL 80% ethanol with subsequent centrifugation for 2 min at 8000× *g* at RT and subsequent drying by centrifugation for 5 min at 8000× *g* at RT. Afterwards, the miRNA fractions were eluted in 14 μL water and quantified at 260 and 280 nm on a TecanM200Pro spectrometer (Tecan Group Lt., Männedorf, Switzerland). Next, 10 ng miRNA (naturally occurring as nonpolyadenylated) were polyadenylated by a poly(A) polymerase and reverse transcribed into cDNA using the miRCURY LNA RT Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Additionally, as a reverse transcription efficiency control, a spike-in control consisting of a synthetic RNA target (Qiagen, Hilden, Germany) was added to the reaction buffer. The reverse transcription step lasted for 60 min at 42 ◦C, followed by an inactivation step for 5 min at 95 ◦C. Quantitative real-time Polymerase Chain

Reaction (qPCR) on pre-designed PCR panels with Locked Nucleic Acids (LNA)-enhanced oligonucleotides (miRCURY LNA miRNA custom PCR array of 84 liver-specific miRNAs (see Supplementary Materials Table S1 for a list of all miRNAs investigated)) was performed from 62.5 pg cDNA on an ABI 7900HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) in 384-well format using miRCURY LNA SYBR Green Master Mix and ROX reference dye (20×) (Qiagen, Hilden, Germany). The thermal profile was as follows: initiation (2 min, 95 ◦C), denaturation (10 s, 95 ◦C, 40 cycles), annealing/elongation (1 min, 56 ◦C, 40 cycles) and dissociation curve analysis. Results were analyzed using the QIAGEN GeneGlobe miRCURY LNA miRNA PCR Data analysis software. That is, Ct values were evaluated in consideration of an interplate calibrator, RNA isolation efficiency spike-in controls and reverse transcription efficiency spike-in control, and subsequently normalized to the expression of a set of housekeeping genes such as the miRNAs *miR-103a-3p*, *-16-5p*, *-191-5p*, *-423-5p* and *-23a-3p* and a small nucleolar RNA (*snoRD38D*) and referred to the values of the solvent control (2.5% ACN for 8 h and 0.35% ACN for 24 h), which is in accordance with the 2−ΔΔCt method [40].
