Cells were maintained at 37°C and 5% CO2 for 0-24 hours. Notably, all cell-culture experiments using LC3 western blotting (a protein central to autophagosome formation) as an endpoint were performed with the addition of the protease inhibitors, E-64d (10 mg/mL; Enzo Life Sciences) and pepstatinA (10 mg/mL; Sigma). Cells were then either harvested for protein extraction or fixed to coverslips with paraformaldehyde for immunohistochemistry. Animal protocols were approved by the University of Pittsburgh

Institutional Animal Care and Use Committee. Experiments CH5424802 price were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals. Cecal ligation and perforation (CLP) was performed on C57BL/6 male mice (Jackson Laboratories, Bar Harbor, ME) 6-8 weeks in age and weighing 20-25 g. These animals were anesthetized with pentobarbital (70 mg/kg, intraperitoneal [IP]). A 1- to 2-cm midline laparotomy was performed, and the cecum was identified. The stool was then manipulated to the tip of the C59 wnt clinical trial cecum and was subsequently ligated 1 cm from the tip with a 2-0 silk tie. The cecum was then perforated with a 22-gauge needle and returned into the abdomen. The muscle and skin were closed with a running 2-0 silk suture. Sham-operated animals underwent laparotomy

and bowel manipulation without ligation or perforation. Tissue and blood collection occurred at either 8 or 20 hours post-CLP. No antibiotics were used,

and animals had free access to food and water pre- and postoperatively. HO was inhibited in vivo through an IP injection of SnPP (50 mg/kg) 1 hour before CLP or with the use of in vivo HO-1–specific siRNA (50 μM/kg) (Invitrogen). This was administered via hydrodynamic tail vein injection, where the siRNA was made to the correct concentration in 2 mL of lactated ringers and given 3 days before CLP. The rapid injection of this large volume creates significant pressure to help promote siRNA uptake. Scrambled siRNA (50 μM/kg) was used as a control again via hydrodynamic tail vein injection. Autophagy was inhibited selleck kinase inhibitor through the use of in vivo siRNA against VPS34 (50 μM/kg; Invitrogen). p38 MAPK was inhibited in vivo through IP injections of SB203580 (10 mg/kg; Calbiochem) 1 hour before CLP. Cells were fixed on coverslips with paraformaldehyde for 15 minutes and then rinsed with cold phosphate-buffered saline (PBS). Slides were then stained for LC3 (Novus) to monitor autophagy. Liver tissue from mice was removed after perfusion with cold PBS and paraformaldehyde. Tissue was then placed in paraformaldehyde for 1 hour, then switched to 30% sucrose solution for 12 hours. The tissue was then slowly frozen in 2-methylbutane. Sections were stained using antibodies against LC3, HO-1 (Enzo Life Sciences), VPS34 (Invitrogen), and phosphorylated p38 MAPK (Cell Signaling). Images were taken with a Zeiss 510 inverted confocal microscope.