*2.4. RNA Isolation and qPCR Analysis*

To investigate the deregulation of gene expression of the selected liver-specific miR-NAs, HepaRG cells were seeded, cultivated and adapted to treatment medium as described in Section 2.3. The cells were incubated with 35 μM Sc for 2, 4, 6, 8 and 24 h, respectively. The mRNA and miRNA were isolated using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, including a purification step of miRNA-enriched fractions using the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany). Cell lysis and total RNA isolation occurred as mentioned in Section 2.3. with the exception that no spike-in control was added. Additionally, the RNeasy Mini spin columns containing the mRNA fractions were not discarded, but stored at 4 ◦C until further isolation. The flow-through containing the smaller miRNA fractions was purified and miRNA eluted as mentioned in Section 2.3. Afterwards, mRNA isolation occurred as follows: washing of RNeasy Mini columns once with 700 μL RWT buffer and twice with 500 μL RPE buffer with centrifugation for 15 s at 8000× *g* at RT between each step, respectively, and drying by centrifugation for 1 min at full speed at RT. The samples were eluted in 30 μL water. Both the miRNA enriched samples and the total RNA samples were quantified at 260 and 280 nm on a TecanM200Pro spectrometer (Tecan Group Lt., Männedorf, Switzerland).

Five hundred ng miRNA were polyadenylated and reverse transcribed into cDNA using the miScript II RT Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The cDNA was synthesized by using a poly(T) primer with a 5 universal tag, enabling the alignment with a universal primer during qPCR amplification. qPCR was performed from 227.3 pg cDNA on an ABI 7900HT Fast Real-Time PCR system in 384-well format using miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany) with 10x universal primer and 10x miRNA-specific miScript Primer Assay (Qiagen, Hilden, Germany). The thermal profile was as follows: initial activation (15 min, 95 ◦C), 3-step cycling (40 cycles in total) with denaturation (15 s, 94 ◦C), annealing (30 s, 55 ◦C), elongation (34 s, 70 ◦C), and dissociation curve analysis. Ct values were evaluated according to the 2−ΔΔCt method [40] by normalizing the respective Ct-values to the expression level of miR-103a and referring to the solvent control (0.35% ACN).

One microgram of total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. Using 20 ng cDNA, qPCR was conducted on an ABI 7900HT Fast Real-Time PCR system in 384-well format with Maxima SYBR Green/ROX qPCR Mastermix (Thermo Fisher Scientific, Waltham, MA, USA) and 300 nM of each primer (synthesized at Eurofins Genomics, Ebersberg, Germany, see Table 1). The thermal profile was as follows: initial denaturation (15 min, 95 ◦C), 3-step cycling (40 cycles in total) with denaturation (30 s, 95 ◦C), annealing and elongation (1 min, 60 ◦C), final elongation

(10 min, 60 ◦C), and dissociation curve analysis. Ct values were evaluated according to the 2−ΔΔCt method and normalized to the housekeeping gene *GUSB* (β-glucuronidase).

**Table 1.** Sequences of primers used for qPCR analysis of target genes.


#### *2.5. Ingenuity Pathway Analysis*

The target genes of miRNAs with significantly altered expression values in HepaRG cells after treatment with 35 μM Sc for 24 h were predicted with Ingenuity Pathway Analysis software (IPA, version 70750971, Qiagen Bioinformatics, Redwood City, CA, USA). IPA uses experimentally validated targeting interactions from TarBase [41], miRecords [42] and Ingenuity expert findings, and predicted miRNA–mRNA interactions from TargetScan [43]. The predicted target genes were compared to the gene expression dataset from a whole genome microarray conducted with primary human hepatocytes incubated with 100 μM Sc for 24 h [44]. The matched targets were prioritized according to their miRNA and mRNA relationship. This included opposing expression pairing and "highly predicted" and/or "experimentally observed" confidence of miRNA-target gene correlation. Subsequently, IPA core analysis and expression analysis with the matched target genes were performed to predict affected diseases and functions and to determine possible signaling pathways involved in PA toxicity.

### *2.6. AntagomiR Experiments*

In order to identify the targets of the miRNA-4434 (also known as human miRNA-4516) and to assess if this specific miRNA expression is sensitive to PA exposure in HepaRG cells, so-called antagomiR experiments were conducted. For this purpose, HepaRG cells were seeded, cultivated and adapted to treatment medium as described in Section 2.3. The cells were incubated with 35 μM Sc and transfected with Lipofectamine RNAiMAX (Thermo Fisher Scientific Waltham, MA, USA) reagent with either miRCURY LNA miRNA Power Inhibitor (antagomiR)-4434 (5- -3 sequence: TTCTACTTTACTTCTCCT, matches miRNA-4434 in its seed region GGAGAAG), miScript Inhibitor Negative Control (5- -3 sequence: TAACACGTCTATACGCCCA; both from Qiagen, Hilden, Germany) as an antagomiR-Negative Control (antagomiR-NC) or water as a negative treatment control. RNAiMAX and antagomiR-4434, antagomiR-NC or water were prepared separately in Opti-MEM reduced serum medium before being mixed 1:1 and incubated for 5 min at RT. Afterwards,

the antagomiR-lipid complexes were added to the cells, resulting in a final concentration of 100 pmol (50 nM) or 150 pmol (75 nM) antagomiR-4434 or anagomiR-NC per 6-well, respectively. According to the manufacturer's protocol, antisense effects are usually assessed 24–72 h after transfection. Therefore, in this case, total RNA was isolated 48 h after transfection and isolation was conducted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, as described elsewhere [45]. Quantity and purity of isolated RNA were measured at 260 and 280 nm on a TecanM200Pro spectrometer. Then, 1 μg total RNA was reverse transcribed into cDNA and qPCR was performed from 20 ng cDNA as described in Section 2.4.
