Pkr inhibitor

HepG2 cells (JCRB1054) were purchased from the Health Science Research Resources Bank (Japan Health Sciences Foundation, Osaka, Japan) and maintained at 37 °C under an atmosphere of 95% air and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were immersed in a culture medium containing the indicated concentrations of Zebularine, 5-Aza-2′-deoxycytidine (5-aza-dC), 2-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-3-(1H-indol-3-yl) propionic acid (RG108) or PKR inhibitor. Zebularine (Wako Pure Chemical Industries, Osaka, Japan) was dissolved in distilled water as a stock solution. 5-Aza-2′-deoxycytidine, RG108 (Wako Pure Chemical Industries), PKR inhibitor (Merck Millipore, Tokyo, Japan), acetaminophen (Sigma-Aldrich Japan, Tokyo, Japan) and aflatoxin B1 (Wako Pure Chemical Industries) were each dissolved in DMSO as a stock solution.

Lipopolysaccharides (LPS; Merck) at a concentration of 100 ng/mL or tumor growth factor-β (TGF-β; Merck) at a concentration of 3.5 ng/mL were added to the DMEM and the cells were cultured for 24 hours for LX-2 cell (1.5×10⁵ /well) activation. Separately, palmitic acid (200 nM) (Merck) was added to the DMEM and the cells were cultured for six hours for LX-2 cell activation. For inhibiting activation of LX-2 cells, 200 nM C16, a PKR inhibitor (Merck) or 100 μM JNK inhibitor Ⅱ (Merck) or 40 μM U0216, a MEK inhibitor (Merck) was supplied one hour before addition of LPS or palmitic acid.

In vitro cell viability was quantified with the MTS assay (Promega, Fitchburg, WI, USA). Huh7 cells treated with several concentrations (500–3000 nM) of the PKR inhibitor (Merck) were seeded in 96-well plates at 2 ×10³ cells per well. At each time point, cells were treated with MTS reagent and incubated for 120 min. Absorbance at 450 nm was recorded. Cells treated with DMSO were used as controls. The wells were photographed under the microscope.