AML-12 cell culture.

The murine hepatocyte cell line, AML-12, was purchased through the American Tissue Culture Collection (ATCC) and grown in DMEM:F12 Medium supplemented with 10% FBS, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium (Invitrogen), and 40 ng/mL dexamethasone (MilliporeSigma) (complete medium). For experiments, cells were acclimated to complete medium without dexamethasone for 18 hours prior to stimulation with bacterial LPS (Thermo Fisher Scientific). AML-12 cells were treated with 50 μM MIF098 (45), ERK activation inhibitor U0126 (Cell Signaling Technologies), or vehicle control (0.1% DMSO; VWR Chemicals LLC) 1 hour prior to LPS challenge at the indicated concentrations. RNA was isolated with the Direct-zol RNA Kit (Zymo Research), and protein was isolated as previously described (14). Cell lysates were separated on 10% polyacrylamide gels and used for Western blot analysis with antibodies against phospho-ERK1/2 (sc-7383, Santa Cruz Biotechnology Inc.), phospho-p65 (3033S, Cell Signaling Technology), total ERK (06-182, MilliporeSigma), total p65 (6956S, Cell Signaling Technology), and IκBα (9242, Cell Signaling Technology). GAPDH (MAB374, MilliporeSigma) was used as a loading control. Signal intensities were quantified using ImageJ (NIH).