Fifty mg selenite / 100 g body weight was administered by way of drinking water. In the promotion study, selenium exposure started 1 week before check details 2-AAF feeding until sacrifice at days 7 and 21 post-PH. In the progression study, selenium exposure was for 3 months starting 3 weeks after PH. Primary human hepatocytes were obtained
from LONZA (Basel, Switzerland). Primary rat hepatocytes were isolated.26 HCC-1.2 and HCC-3 cell lines were established in our laboratory27; SNU398 cell line was purchased from ATCC (LGC Standards, Wesel, Germany). The cell lines were kept under standard tissue culture conditions. Fifty nM of sodium selenite (Sigma-Aldrich) was added 24 hours before treatment. Synthesized linoleic acid hydroperoxides (LOOH)28 was dispersed by sonication into serum-free medium containing 1 mg/ml fatty acid-free bovine serum albumin (BSA). ROS was quantified by the 2′,7′-dichlorofluorescin diacetate (DHFC) method.21 LOOH-Ab Fostamatinib were detected in plasma according to the modified method of Rolla et al.29; 1 mM DTPA (Sigma-Aldrich) was added to washing phosphate-buffered saline (PBS) (Invitrogen, Carlsbad, CA). HCC tissue arrays were
stained for c-jun by immunohistochemistry, counterstained with hematoxylin, and scanned using TissueFaxs software (TissueGnostics, Vienna, Austria). Nuclear localization of c-jun was evaluated using HistoQuest software (TissueGnostics). Proper recognition of nuclei by the hematoxylin nuclear mask was confirmed prior to quantification of c-jun nuclear intensity. RNA was isolated according to a standard Trizol-extraction protocol (Invitrogen, Austria). Complementary DNA (cDNA) was synthesized using High Capacity cDNA Reverse Transcription
Kit (Applied Biosystems, Foster City, CA) and assessed for gene expression with the real-time RT-PCR TaqMan System using the following primers: Hs00173626_m1 for VEGF, Hs00174103_m1 for IL-8, Hs01591589_m1 for GPx2, and Hs00157812_m1 for MCE Gpx4 (Applied Biosystems). The ΔΔCt method was applied for quantification. Total GPx activity in cell lysates was measured as described.30 Western blotting was performed as described.31 More details are given in the Supporting Materials. Human serum VEGF and IL-8 were determined by Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Abingdon, UK) and IL-8 Human ELISA Kit (BenderMedSystems, Vienna, Austria), respectively, according to the manufacturers’ instructions. AP-1 and HIF-1 DNA binding was measured in nuclear extracts by TransAM transcription factor ELISA (Active Motif Europe, Rixensart, Belgium) according to the Instruction Manual. All cellular experiments were performed at least three times.