Hepa1 6 cell line

Male C57BL/6 (B6) and BALB/c mice were purchased from the National Laboratory Animal Center (Taiwan). IL-6 knockout mice on a C57BL/6J background (B6.129S2-Il6tm1Kopf/J) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All animal experiments were approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital (IACUC permit number: 2014123001, date of approval on 1 March 2015). The Hepa 1-6 cell line, which is a mouse hepatoma cell line derived from ATCC CRL-1830 and obtained from the Bioresource Collection and Research Center (Hsinchu city, Taiwan), was used in this study.

Experiments were performed using the Hepa1-6 cell line (# CRL-1830, ATCC). Cells were cultured in DMEM media with 10% fetal bovine serum (FBS), 1 g/L glucose, 2 mM glutamine, 100 units/mL penicillin, and 100 mcg/mL streptomycin and 30 μM oleic acid. After seeding, 30,000 cells per well in 96 well plates, and cells were incubated at 37 °C with 5% CO2 overnight to let the cells attach to the culture surface. Mitochondrial membrane potentials (μM) were assessed with a Tetramethylrhodamine (TMRE) fluorophore (Thermo Fisher Scientific, Waltham, MA, USA, # T669). Cells were treated with SIMR1281 for 24 h. Then, TMRE was added to the cells at 200 nM and incubated for 45 min at 37 °C. In separate control wells, incubation with FCCP (10 µM) for 30 min before the addition of TMRE was used as a positive control. Cells were washed once with PBS, and a fresh culture medium was added before measurement. TMRE fluorescence was measured at Ex/Em = 545/580 nm. Signals in the vehicle-treated and blank-well controls were used to normalize the results.