Ten-minute static planar images were acquired with a 256 pixel × 256 pixel matrix. The liver area (mm2) was determined by the amount of radioactivity uptake in the organ. Neutrophil accumulation in the liver was quantified with MPO activity assays as previously described.22 MPO activity was assayed with measurements of the variation in the optical

density (OD) at 450 nm with tetramethylbenzidine (1.6 mM) and hydrogen peroxide (0.5 mM). The results are expressed as relative neutrophil numbers, and they were calculated by comparisons of tissue supernatant OD values with the OD values of a standard neutrophil curve (>95% purity). The results are expressed as means ICG-001 and standard errors of the mean. Prism (GraphPad Software, San Diego, CA) was C59 wnt manufacturer used for data analysis. Statistical significance was tested with the Student t test or a one-way analysis of variance followed by Bonferroni posttests, and P < 0.05 was considered to indicate statistical significance. PV fused to an MTS and GFP was developed as a genetically encoded Ca buffer. GFP targeted to the mitochondrial matrix was used as a control (Fig. 1A). PV was effectively expressed in SKHep1 cells transfected with PV-MITO-GFP, as demonstrated by immunoblotting (Fig. 1B). Moreover, PV was correctly targeted to the mitochondrial matrix, as demonstrated by the colocalization of GFP and MitoTracker Red

(Fig. 1C). For the evaluation of Ca2+ buffering by this construct, SKHep1 cells were stimulated with 1 μM ATP, and Ca was measured by Rhod-2/AM confocal microscopy. ATP elicited a robust increase in Ca in control cells and in cells expressing GFP alone, but this was reduced by approximately 90% in cells expressing PV in mitochondria (n = 3, P < 0.001; Fig. 2). These results

demonstrated that PV-MITO-GFP was correctly targeted to the mitochondrial matrix and efficiently buffered agonist-induced Ca signals. Ca plays a crucial role in apoptosis, so we investigated the effect of Ca buffering on cell death. A treatment with STA increased the percentage of dead cells to 19.1% ± 3.7% (11.4% ± 0.7% for unstimulated cells, P < 0.001, n = 3). Upon Ca buffering, the rate of cell death induced by STA was reduced to 7.7% ± 2.2%, whereas the rate of cell death remained high (25.7% ± 1.8%) MCE in cells transfected with MITO-GFP (P < 0.001, n = 3; Fig. 3A). The role of Ca in cell death was further characterized by the evaluation of the intrinsic or extrinsic apoptotic pathways because the two pathways converge at the level of Ca signaling.23 The intrinsic pathway was investigated through the measurement of caspase-9 and caspase-3 activity in SKHep1 cells stimulated with 100 nM STA for 6 hours. Caspase-9 activity was increased to 0.16% ± 0.06% after the STA treatment compared with 0.1% ± 0.02% in control cells, and this was blocked by the expression of PV-MITO-GFP (P < 0.001, n = 3; Fig. 3B). Similarly, STA-induced caspase-3 activity was inhibited by Ca buffering (Fig. 3C). Caspase-3 activity was increased from 43.