*2.7. Cell Viability Assay*

In order to exclude antagomiR-mediated cytotoxicity on PA-incubated HepaRG cells, cells were seeded at a density of 9000 cells per well in the inner 60 wells of a 96-well plate and cultivated as described in Section 2.2. After differentiation, cells were incubated with 35 μM Sc and transfected with either antagomiR-4434 or antagomiR-NC at concentrations of 25, 50, 75 and 100 nM, respectively, as described in Section 2.5. Transfection reagent with water was used as a negative control and 0.01% Triton-X-100 served as positive control for cytotoxicity. The Neutral Red Uptake (NRU) assay was applied for assessing the cell viability. Briefly, 120 μL 3-amino-7-dimethylamino-2-methylphenazine hydrochloride (neutral red) reagent dissolved in cell culture medium (40 μg/mL) was added per well and incubated for 2 h at 37 ◦C. Afterwards, the supernatant was discarded, cells were washed twice with Phosphate Buffered Saline (PBS) and dye crystals dissolved in 150 μL desorption solution (1% acetic acid in 50% ethanol). After shaking for 10 min (under protection from light), fluorescence was measured at λex = 530 nm and λem = 645 nm on a TecanM200Pro spectrometer.
