Subsequent breeding yielded genotypes for experiments

Bax-YFP fusion construct into mitochondria. Results demonstrate that such treatment decreased the re-localization of Bax from the cytosol to the mitochondria, as measured by the YFP-Bax fluorescence assay. We excluded any inhibitory role of NAC on the transcription activity of E2F1 by confirming that the transcription levels of E2F reported construct B-Mybluciferase did not change after NAC treatment. These results strongly imply that production of ROS is essential for the apoptotic effect of E2F1 in PC12 cells and suggest that generation of ROS could be the initiation event for the apoptotic response. Next we investigated whether this finding could be extended to other neuron cell lines. To this end, we stable transfected SHSY5Y and SK-N-JD neuroblastoma cells with ER-E2F1 expression vector and measured apoptosis and ROS production after the addition of OHT. As shown in Fig. 6A, the presence of OHT increased caspases-3 activity as well as ROS production in SHSY5Y cells. In contrast in SK-N-JD cells, activation of E2F1 by the addition of OHT did not affect ROS production, and interestingly, there was no effect on apoptosis. This was not due to a failure to induce E2F1, as immunofluorescence studies show that the addition of OHT induced the translocation of ER-E2F1 to the nuclei equally in both cell types. Likewise, co-transfection of both cell types with ER-E2F1 and an E2F-luciferase reporter shows that OHT induced E2F1 transcriptional activity to a similar extent. These data demonstrate that the differential 10 E2F1 and ROS 1022150-57-7 chemical information ability of E2F1 to induce apoptosis in SH-SY5Y versus SK-N-JD cells correlates with its ability to generate ROS. E2F1 Regulates the Expression of a Large Number of Genes Involved on ROS Metabolism The steady-state levels of ROS are controlled by the rate of ROS production and clearance by scavenging mechanisms. Expression levels were compared between with and without OHT addition. Hypoxanthine phosphoribosyltransferase 1 gene was used as control for each gene expression calculation, and the extent of change in the expression of each gene was calculated by the DCt method. Only genes whose expression was downregulated at least 2 fold are shown in the primarily generated following release of electrons from the electron transport chain at the mitochondria, NOXs are capable of generating ROS in a highly regulated manner. In order to investigate whether the intracellular accumulation of ROS after induction of E2F1 is due to the upregulation of NOX, we have studied the effect of NOX-inhibitor diphenyleneiodonium on the E2F1-induced apoptosis by measuring caspase-3 activation. As shown in Fig 7A, DPI treatment repressed caspase-3 activity in the absence or presence of OHT, however there were significant differences in the extent of caspase-3 repression. Similarly, DPI treatment only led to partial diminishing of ROS levels in the presence of OHT as shown in Fig 7B. The inability of DPI to impair ROS generation, suggests that E2F1 regulates ROS production by mechanisms distinct from the NOXs. As previous reports demonstrated that E2F1 represses the expression of antioxidative enzyme Mn superoxide dismutase through inhibition of NF-kB, we next evaluated whether regulation of MnSOD was implicated in the E2F1induced apoptotic response. To test this possibility, the expression of MnSOD and mRNA levels were determined at different times following OHT treatment, with CycE mRNA expression used as a positive control. As shown in Fi