The amount of total DNA transfected was equalized with the appropriate amounts of control vectors

n X-100 in saline. LDH activity present in the culture media, as well as LDH activity present in lysates was measured spectrophotometrically at 490 nm on a 96-well plate reader using the Cytotox 96 Kit as previously described. Cellular death was expressed as the percentage of LDH released. Extraction of mitochondrial and cytosolic fractions Cells were grown in 6-well culture plates until 80% confluence was reached. Next, mitochondrial and cytosolic fractions were extracted as previously described. Briefly, cells were treated with vehicle alone or in combination with MnTBAP or SN-50 for 24 and 48 h. Afterwards, cells were washed twice with PBS, scraped and collected by centrifugation at 1 5006g for 10 min. Cell pellets were resuspended in 200 mL of extraction buffer and homogenized with a pellet pestle . Homogenates were maintained on ice for 15 min and then centrifuged at 8006g for 5 min. The pellet, containing the nuclei and whole cells, was discarded and the supernatant was centrifuged at 20 0006g. The supernatants, i.e. cytosolic fractions, were removed and stored at 280uC until analyzed by gel electrophoresis. Pellets containing mitochondria were resuspended in 50 mL of extraction buffer, homogenized with a pestle and then centrifuged at 20 0006g. The supernatants, i.e. mitochondrial fractions, were removed and analyzed by gel electrophoresis. DNA fragmentation analysis Cells were grown in a 25 cm culture flask until 80% confluence was reached, and they were then treated with vehicle or AAP. Forty-eight hours later, cells were collected by scraping and centrifuged at 8006g for 10 min. Pellets were washed twice with PBS-MgCl2 and then resuspended in lysis buffer containing 0.125% proteinase K and maintained at 50uC overnight. After centrifugation at 10 0006g, fragmented DNA in the supernatant was extracted by adding a mixture of phenol/ chloroform/isoamyl alcohol and centrifuged at 10 0006g. Fragmented DNA in the aqueous phase was precipitated by adding sodium acetate and absolute ethanol and then isolated by centrifugation at 10 0006g for 20 min. The DNA pellet was dissolved in 25 mL of a 10 mM TrisHCl, pH 7.4 solution containing 1 mM EDTA. DNA samples 2 Extraction of nuclear and cytosolic fractions Cells were grown in 6-well culture plates until 80% confluence was reached. Next, cells were treated with vehicle or AAP alone or in combination with MnTBAP or SN-50 for 18 h. After the incubation period, cells were washed twice with PBS, scraped and collected by centrifugation at 1 5006g for 10 min. Cell pellets were resuspended in 200 mL extraction buffer A and incubated for 15 min on ice. Afterwards, Nonidet P-40 was added and samples were vortexed for 30 sec at 4uC. After centrifugation at 10 0006g for 1 min at 4uC supernatants, i.e. cytosolic fractions, were removed and stored at 280uC until analyzed by gel electrophoresis. Pellets containing nuclei were resuspended in 50 mL of extraction buffer C and nuclear proteins were extracted by shaking the samples for 30 min at 4uC. Afterwards, samples were centrifuged at 20 0006g for 5 min at 4uC. The supernatants, i.e. nuclear fractions, were removed and analyzed by gel electrophoresis. fluorescent probe CM-H2DCFDA from Molecular Probes and the HRP-conjugated IgG antibodies from Chebulinic acid DakoCytomation S.A.. The ELISA Kit for IL-1b detection was from RD systems. All other reagents were obtained from Sigma-Aldrich. Western blot analysis Immunoblot analysis was performed as previously described, on c