Fifty AZD5363 clinical trial microliters of tissue extract was used for GSH measurement. GSH was measured according to the manufacturer’s instructions with a GSH assay kit (Promega, Madison, WI). Luminescence was detected with a Synergy 2 multimode microplate reader with Gen5 data analysis software (BioTek, Winooski, VT). DNA was extracted from 25 mg of mouse liver. Total DNA purification was performed with a DNeasy blood and tissue kit (Qiagen, Germantown, MA) according to the manufacturer’s instructions. RNA was eliminated by incubation in 5 μg/mL RNase (Roche, Indianapolis, IN). A 1.5-μg DNA aliquot was loaded onto 1.5% agarose gel for separation to assess for DNA fragmentation.

TUNEL staining was conducted with an in situ apoptosis detection kit from R&D Systems according to the manufacturer’s instructions. Six to seven animals were Osimertinib purchase used per time point per treatment group. At least 1000 cells (TUNEL-positive cells and TUNEL-negative cells) were counted in each of eight separate low-power fields for each animal, and the percentage of TUNEL-positive cells was calculated. Two hours prior to sacrifice, animals received 30 μg of BrdU/g of body weight intraperitoneally. Liver

specimens were obtained, fixed in 4% paraformaldehyde, processed for histological analysis, and stained with the Amersham cell proliferation kit (Amersham Pharmacia Biotech, Ltd., United Kingdom). BrdU incorporation was measured at 24, 36, 48, and 72 hours. There were four to seven mice per treatment group per time point. At least 1000 cells (BrdU-positive cells filipin and BrdU-negative cells) were counted

in each of eight separate low-power fields for each animal, and the percentage of BrdU-positive cells was calculated. Hepatic cytoplasmic and nuclear proteins were extracted with the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s instructions. Liver samples were homogenized in a radioimmunoprecipitation assay buffer (50 mM trishydroxymethylaminomethane–hydrochloric acid, pH 7.4, 150 mM sodium chloride, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, 1 mM ethylene diamine tetraacetic acid, and protease and phosphatase inhibitors). Four hundred micrograms of protein was used for immunoprecipitation. The lysate was precleared with 1 μg of isotype immunoglobulin G (IgG) and 50 μL of protein A/G agarose at 4°C for 1 hour. Five micrograms of an immunoprecipitating antibody or isotype IgG was added to the supernatant, and it was rocked overnight at 4°C. Next, 50 μL of protein agarose was added, and the mixture was rocked for 2 hours at 4°C. The protein bound to the agarose conjugate was centrifuged, and the pellet was washed three times with a radioimmunoprecipitation assay buffer.