Fresh samples

of liver tissue were also collected, immediately frozen in isopentane, and embedded in OCT. Cryosections at 5 μm were stained with oil red-O for evaluation of hepatic steatosis as described7, 14, 18 (see Supporting Experimental Procedures). The detection of F4/80, a specific marker of murine macrophages,19 was performed as described7, 18 with slight modifications (see Supporting Experimental Procedures). Serum biochemistry and total hepatic triglyceride content was determined by standard laboratory procedures (see Supporting Experimental Procedures). Cleaved caspase-3 detection in samples of liver tissue was performed KU-60019 solubility dmso by immunohistochemistry as described in the Supporting Experimental Procedures. Mouse hepatocytes were isolated from 21-week-old WT (n = 12), ApoE−/− (n = 4) and ApoE−/−/5-LO−/− (n = 4) mice. Animals were anesthetized with ketamine/xylazine and liver cells isolated by in situ collagenase perfusion through the portal vein as described with modifications10, 11 (see Supporting Experimental Procedures). Caspase-3/7 activity was determined with the Caspase-Glo 3/7® Assay (Promega, Madison, WI). Isolated hepatocytes were seeded at a density of 30,000–40,000 cells/well in white-walled 96-well plates and incubated for 12 hours with vehicle (<0.02% ethanol), TNF-α (20 ng/mL) EPZ-6438 ic50 and/or actinomycin D

(50 ng/mL). Hepatocytes were also exposed to increasing concentrations (0.01 and 0.1 μM) of LTB4, LTD4, or 5-HETE in the absence or presence of TNF-α (20 ng/mL) and/or actinomycin D (50 ng/mL). In some experiments, hepatocytes were pretreated for 30 minutes with 1 μM of selective LTB4(U-75302) and LTD4 (MK-571) receptor antagonists (Cayman Chemical, Ann Arbor, MI). At the end of the incubations, the luminogenic caspase 3/7 substrate was added and caspase-3/7 activity was assessed by measuring the luminescence

signal in a microplate luminometer FluoStar Optima (BMG Labtech, Offenburg, Germany). SDHB Total RNA was obtained from liver and adipose tissue with the RNAqueous kit (Ambion, Austin, TX) and the Trizol reagent (Invitrogen, Carlsbad, CA), respectively. RNA concentration was assessed in an ultraviolet spectrophotometer, and its integrity was tested in a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Samples were retrotranscribed with the high-capacity complementary DNA archive kit (Applied Biosystems, Foster City, CA). Quantitative analysis of gene expression was performed by real-time polymerase chain reaction (PCR) in an ABI Prism 7900 Sequence Detection System (Applied Biosystems). Ready-to-use primer and probe sets were used as described in the Supporting Experimental Procedures. NF-κB activity was assessed in nuclear extracts from liver tissue and isolated hepatocytes incubated with vehicle (<0.