The active form of STAT3 is quickly translocated to the host cell nucleus

ytosolic, nuclear and mitochondrial fractions from vehicle- and AAP-treated cells. Protein samples were loaded on 15% PAGE-SDS gels and transferred onto nitrocellulose membranes. Membranes were blocked in PBS-Tween 20 containing 5% non-fat dry milk and 0.1 % BSA for 1 h at 4uC and then incubated with either polyclonal anti-cytochrome c antibody, monoclonal anti-Bax antibody, monoclonal anti-NF-kB p65 antibody, monoclonal antiphospho-NFkB p65 antibody; polyclonal atubulin antibody, monoclonal anti-H2A antibody or monoclonal anti-OxPhos Complex IV subunit IV antibody overnight at 4 uC. Afterwards, blots were washed with PBS-Tween 20 and incubated with HRP-antimouse IgG for 2 h at 4uC. Immunoreactive bands were visualized using an enhanced chemiluminiscence system. Statistical analysis Data are expressed as the mean 6 S.E.M. Statistical analyses were performed using one-way analysis of variance and the a posteriori Bonferroni’s test for multiple comparisons using GraphPad software. p values less than 0.05 were considered statistically significant. Statistical results are reported in the figure legends. Results 3.1. Effect of AAP on SH-SY5Y viability In order to evaluate the effect of AAP on SH-SY5Y human neuroblastoma viability, cells were treated with various concentrations of AAP for 24, 48 and 72 h and the percentage of MTT transformed as well as the percentage of LDH activity released to the culture medium were measured as indices of cellular death. Cells treated with AAP showed a decrease in the percentage of MTT transformed in relation to vehicle-treated cells in a concentration- and time-dependent manner. AAP significantly reduced mitochondrial function 24 h after treatment, reaching a reduction in the percentage of MTT transformed to about 60% of control values 72 h after treatment with AAP. Similarly, AAP induced an increase in the percentage of LDH released in a concentration- and timedependent manner. LDH activity has been considered an index of necrosis or of secondary necrotic cell death after apoptosis occurring in cultures in which the phagocytic component is absent and apoptotic bodies cannot be removed. AAP-treatment caused a loss of cell viability, determined as % LDH released, ranging from 20% to about 30 % 72 h after treatment with AAP 1 mM and 2 mM respectively. Since AAP 2 mM significantly reduced neuroblastoma viability at all times studied, this concentration was selected to perform further experiments to elucidate the molecular mechanism involved. Reactive oxygen species production Cells were grown on poly-L-lysine-coated glass coverslips until 80% confluence was reached, and then cells were treated with vehicle or AAP for 3 h, 6 h, 18 h and 24 h. To monitor reactive oxygen species production, cells were loaded by incubation with CM-H2DCFDA in Krebs-Henseleit solution as described previously. ROS production monitoring was performed at room temperature on the stage of a Nikon Eclipse TE200 inverted microscope equipped with a 75W Xenon lamp and a Nikon 40X, 1.3 numerical aperture, epifluorescence oil immersion objective. Images were acquired with a CCD camera and analyzed using commercial software. Background was subtracted and fluorescence was recorded using an Relebactam excitation filter of 535 nm and an emission filter of 635 nm. Frames were recorded every 15 s over a 10 min period. Linear regression of fluorescence data was obtained for each condition and the slope of the best fitting line was taken as an index of ROS prod