Staurosporine (STA) was acquired from Calbiochem (San Diego, CA). All other reagents were of the highest quality that was commercially available. Male Holtzman rats (40-50 g), which were obtained from CEBIO (Centro de Bioterismo, Federal University of Minas Gerais, Belo GSK2126458 ic50 Horizonte, Minas Gerais, Brazil), were used for all studies. The animals were maintained on a standard diet and were housed with a 12-hour light-dark cycle. The investigation conformed to the standards of Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85-23, 1996 revision). Complementary DNA (cDNA) for the Ca2+ binding protein parvalbumin (PV) was subcloned between the BamHI and AgeI restriction sites

of the pAc1GFP1-Mito vector. The resulting vector encoded PV, which was fused to the mitochondrial targeting

sequence selleckchem (MTS) and green fluorescent protein (GFP), and it was called parvalbumin–mitochondrial targeting sequence–green fluorescent protein (PV-MITO-GFP). A recombinant adenovirus was used to deliver the parvalbumin–mitochondrial targeting sequence–green fluorescent protein construct (Ad-PV-Mito-GFP). The virus was amplified with HEK-293 cells and was purified with the VivaPure AdenoPack kit (Sartorius, Göttingen, Germany) according to the manufacturer’s protocol. pAd-PV-MITO-GFP (3 × 109 pfu) was injected into rats by tail vein infusions, and the livers were processed at the indicated times. Cells were perfused with ATP (1 μM), and Ca was monitored in SKHep1 cells with time-lapse confocal microscopy, as previously described.14 Transfected cells were identified with GFP fluorescence. MitoTracker Red and GFP colocalization images were collected as described previously.14 Protein lysates from SKHep1 cells or the total liver were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membranes. Western blots were developed with the ECL Plus reagent. Densitometry was performed with ImageJ software (National Institutes of Health, Bethesda, MD). For the determination

of the proportion see more of dead cells, control SKHep1 cells and cells transfected with mitochondrial targeting sequence–green fluorescent protein (MITO-GFP) or PV-MITO-GFP were stimulated with 300 nM STA for 6 hours. The cells were trypsinized, fixed in 70% ethanol, and incubated with 0.5 mg/mL propidium iodide (PI). The cells were analyzed for GFP and PI fluorescence with the Becton Dickinson FACSCalibur system. Total RNA was isolated from SKHep1 cells with TRIzol, and cDNA was synthesized with the SuperScript II kit (Invitrogen). DNA templates were amplified by real-time PCR with the StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) and the SYBR Green method.19 β-Actin was used as an internal control to normalize variations in the cDNA content. Experiments were performed in triplicate for each data point.