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tin condensation Inhibitor 5B . Conversely, the 6 OHDA induced chromatin condensation was enhanced by LY294002, which was an inhibitor of PI3 kinase Inhibitor 5C . These outcomes suggest Hedgehog inhibitor that the PI3 kinase Akt pathway is involved in the 6 OHDA induced apoptosis of PC12 cells Effect of pCPT cAMP on 6 OHDA induced caspase activation As the cellular level of p Akt was improved and the 6 OHDAinduced chromatin condensation was suppressed by pCPTcAMP, the effect of pCPT cAMP on the 6 OHDA induced caspase activation was examined. The activation of caspase 3, 8 and 9 by 6 OHDA was suppressed by pretreatment with 100 M pCPT cAMP Figs.
6A, B and C pCPT cAMP did not suppress the mitochondrial membrane depolarization induced by 6 OHDA To investigate the mechanism of apoptosis suppression by pCPT cAMP, the Hedgehog inhibitor effect of pCPT cAMP on the 6 OHDA induced mitochondrial membrane depolarization was examined with microscopic analysis by double staining with Hoechst33342 and JC 1. Interestingly, pCPT cAMP did not suppress the mitochondrial membrane depolarization despite the fact that pCPT cAMP suppressed chromatin condensation in the exact same cells Inhibitor 7, upper and middle panels . Flow cytometric analysis also showed that pCPT cAMP failed to suppress the mitochondrial depolarization induced by 6 OHDA Inhibitor 7, reduce panel pCPT cAMP inhibitable cleavage of Bid and Ac IETD inhibitable activation of caspase 9 by 6 OHDA Cleavage of Bid by caspase 8 has been shown to directly trigger the release of cytochrome c from mitochondria Kluck et al 1999; Li et al 1998; Luo et al 1998 .
Therefore, we studied the effect of 6 OHDA on the cellular level of cleaved Bid. Western blot analysis revealed Fingolimod that Bid was present as a 22kDa protein in intact PC12 cells. 6 OHDA induced cleavage of Bid to form a 15kDa truncated Bid tBid Inhibitor 8A . This Bid cleavage was inhibited by the presence of 100 M pCPT cAMP Inhibitor 8A . Due to the fact 6 OHDA induces the cleavage of Bid and caspase 9 activation, the effect of Ac IETD CHO, which was an inhibitor of caspase 8 on the caspase 9 activation, was examined to confirm whether caspase 8 activation induces the caspase 9 activation. As shown in Inhibitor 8B, Ac IETD CHO significantly suppressed the 6 OHDA induced caspase 9 activation.
These outcomes suggest that 6 OHDA induced caspase 9 activation is probably through caspase 8 activation, cleavage from the Posttranslational modification Bid and cytochrome c release pathway pCPT cAMP inhibitable phosphorylation of p38 MAPK by 6 OHDA A recent study concerning the 6 OHDA induced apoptosis suggested a correlation among the phosphorylation of p38 mitogen activated protein kinase p p38 and the activation of caspase 8 and 9 in dopaminergic neurons Choi et al 2004 . To study the involvement from the p38 MAPK pathway in PC12 cells, the effect of 6 OHDA on the phosphorylation of p38 was examined. 6 OHDA improved the level of p p38 inside a timedependent manner Figs. 9A and B . Moreover, the 6 OHDAinduced p38 phosphorylation was decreased by pCPT cAMP Figs. 9C and D at the exact same dose and time points that inhibited chromatin condensation. 0.
pCPT cAMP Fingolimod did not suppress intracellular superoxide production induced by 6 OHDA The accumulation of ROS has been reported to play an necessary role in the 6 OHDA induced apoptosis Berman and Hastings, 1999; Choi et al 1999; Double et al 1998; He et al 2000; Salinas et al 2003 . To obtain further insight into the mechanism from the intracellular Hedgehog inhibitor generation of ROS, we employed the superoxide mediated oxidation of hydroethidine to ethidium Yamada et al 2003b and directly assessed the relative rate of superoxide Fingolimod anion generation. As shown in Inhibitor 10A, the fluorescence intensity of ethidium was improved by the therapy with 6 OHDA inside a time dependent manner. The boost in fluorescence intensity was observed from 2min soon after therapy with 50 M 6 OHDA Inhibitor 10A . The fluorescence adjust was suppressed by tiron, a scavenger of Hedgehog inhibitor intracellular superoxide Zuo et al 2000 , but not by pCPTcAMP Figs.
10B and C . In addition, tiron also suppressed the 6 OHDA induced p38 phosphorylation, membrane depolarization and chromatin condensation Inhibitor 11 . A greater concentration and longer pretreatment of tiron resulted inside a far more noticeable inhibition from the membrane depolarization and chromatin condensation Fingolimod Figs. 11D and E . These outcomes indicate that the generation of intracellular ROS, probably superoxide, is essential for the 6 OHDA induced apoptosis, and that 6 OHDA induced CsA insensitive mitochondrial membrane depolarization occurred through the nonspecific membrane damage induced by ROS. 3. Inhibitor In the present function, we demonstrated that 6 OHDA induced apoptosis was dependent on superoxide production, and was inhibited by pCPT cAMP in PC12 cells. The reduce in mitochondrial membrane potential was not inhibited by pCPT cAMP and was not most likely to be involved in the apoptosis machinery in this model. It has been reported that 6 OHDA induces MPT in isolated brain mitochondria Kim et