17 Total RNA was extracted from 106 HepaRG cells with the SV total RNA isolation system (Promega, Madison, WI). Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed using an SYBR Green mix.10 Primer sequences are listed in Table 1. Experimental conditions are given in the Supporting Materials and Methods as described.18 Total cellular protein extracts were obtained by way of cell lysis. Fifty micrograms of protein underwent electrophoresis, and immunoblotting was performed with anti-ADFP (Abcam,

learn more Cambridge), anti-PPARG (Dharmacon), anti-CYP3A4 (Millipore), anti-CYP2E1 (Oxford Biomedical Research), and anti-HSC70 (Tebu-bio) antibodies. Results are expressed as the mean ± SD of three independent experiments. The Mann-Whitney U test was applied to compare data between drug-treated and corresponding control cultures. Data were considered

significantly different at P < 0.05. Preliminary experiments were performed to compare toxic effects of tetracycline and amiodarone after acute and repeat treatments to select several nontoxic and subtoxic concentrations of each drug for further studies (Supporting Results and Supporting Fig. 2). Thus, HepaRG cells were exposed to 50 μM tetracycline, CDK inhibitor 20 μM amiodarone, and 500 μM oleic acid (a positive in vitro steatosis inducer19, 20) for 24 hours or 14 days and stained with Oil Red O to detect intracytoplasmic lipid droplets. Oleic acid induced formation of droplets in hepatocyte-like cells after both acute and repeat incubation, with the vesicles being enlarged after 14 days (Fig. 1c,d). Similarly, lipid vesicles were also observed

in hepatocyte-like cells treated with tetracycline (Fig. 1e,f), but staining was more important after 14 days than 24 hours. Amiodarone also caused accumulation of Oil Red O–stained vesicles in hepatocyte-like cells, but only after 14 days (Fig. 1h). In addition, Thymidine kinase numerous small unstained vesicles were observed in both HepaRG cell types after either 24-hour or 14-day amiodarone treatment, suggesting phospholipid accumulation (Fig. 1g,h). Electron microscopic detection of intracytoplasmic lamellar bodies is thought to be the most reliable method for the identification of phospholipidosis. Exposure of HepaRG cells to 20 μM amiodarone for either 24 hours or 14 days led to the formation of typical concentric lamellar structures corresponding to excessive intracellular accumulation of phospholipids in lysosomes of both hepatocyte-like and biliary-like cells. These lamellar bodies appeared larger after 14-day repeat treatments and mixed with clear vesicles typical of lipid droplets (Fig. 2c,d). As expected, no lamellar bodies were found in tetracycline-treated HepaRG cells (Fig. 2b). Only lipid droplets were observed; they were already detected after 24 hours and were enlarged after 14 days (Fig.