16 μg/g body weight) diluted in sterile saline. The mice were monitored for up to 24 hours, and the time of death was recorded. The Fas injury model was induced in controls and ILK KO mice with a single intraperitoneal injection of Jo-2 at the dose of 0.16 μg/g weight. At the indicated time

points (up to 12 hours) after Jo-2 injection, mice were sacrificed. Livers were snap frozen in liquid nitrogen or formalin-fixed and paraffin embedded for histopathological studies. All procedures performed on these mice were approved under www.selleckchem.com/products/xmu-mp-1.html the IACUC protocol and conducted according to National Institute of Health guidelines. Isolation, culture and treatment of mouse hepatocytes Hepatocytes were isolated from male ILK KO and control mice as described previously [10]. Cells were plated onto collagen-coated 6-well dishes (type I collagen, Collaborative Biomedical, Bedford, MA) 5 × 105 cells per well. Cultures were maintained in minimal essential medium supplemented with 10% fetal calf serum, nonessential amino acids, 2 mM glutamine, and antibiotics (all from Invitrogen). After 2-h incubation medium was removed, and cells were refed the same medium with 0.5% fetal calf serum and incubated overnight. Apoptosis was induced in cultured mouse hepatocytes by treatment

with 0.5 μg/ml anti-Fas antibody and 0.05 μg/ml actinomycin D as described before [12]. The effect of ILK deletion on Fas-mediated apoptosis was also tested in the presence of the extracellular-regulated kinase 1/2 inhibitor U0126 (20 μM, Cell Signaling), the phosphatidylinositol buy C59 wnt 3-kinase (PI3K) inhibitor LY-294002 (50 μM, Cell signaling) and NFκB peptide (30 μM, Calbiochem). Doses of the inhibitors and peptides were selected based on previous studies with isolated hepatocytes [13]. Measurement of apoptosis Apoptotic nuclei were

detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining using the ApopTag Peroxidase kit (Millipore, Billerica, MA). MK-8776 research buy Activation of caspase 3/7 in cell lysates was detected using a commercially available kit (Promega, Madison, WI). Western blot analysis Liver Homogenates were prepared as described previously [10]. The following primary antibodies were Pyruvate dehydrogenase used in this study: rabbit anti-cleaved caspase 3, Rabbit anti-BAD and phospho BAD, Rabbit anti-Bcl-2, Rabbit anti-Bcl-xl, Rabbit anti phospho Akt (serine 473), Rabbit anti phospho ERK (Thr202/Tyr204), Rabbit cleaved PARP, Rabbit p65 (Cell Signaling Technologies, Danvers, MA), Mouse anti Fas (Santa Cruz) and mouse anti-β-actin (Chemicon, Temecula, CA). Donkey anti-rabbit and anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) and were used at 1:50,000 dilutions.