Cell survival was calculated by platting performance as described above. Propidiumiodide discoloration and ROS production were monitored by flow cytometry. Labelling with PI was performed by incubating 106 cells in culture medium containing 2 ug/ml of PI for 15 min. ROS creation was monitored in cells protecting plasma membrane integrity by double staining with dichlorodihydrofluorescein diacetate and PI. Conversion of H2DCFDA to DCF was analysed in PI negative cells. About 106 cells were incubated in culturemediumcontaining 40 ug/mlH2DCFDAfor 45 min at 30 C. 2 ug/ml of PI was added after 30min of incubation. Flow cytometric Dalcetrapib structure analysis was done within an Epics XLTM flow cytometer built with an ion laser emitting a 488 nm beam at 15mW. Natural fluorescence was collected via a 488 nm blocking filter, a nm long pass dichroic and a 525 nm band pass filter. Red fluorescence was obtained by way of a 560 nm small pass dichroic, a nm longpass, and another 670 nmlong pass filter. 20,000 cellswere analysed per sample at low flow rate. Data were analysed by WinMDI 2. 8 software. Cells expressing PKC, Bax c myc, PKC and Bax c myc or none of the proteins were co transformed with pCLbGFP. Cells Gene expression were obtained at different times and fragmentation of the mitochondrial system considered by epifluorescence microscopy. At least 150 cells per sample were classified. In this set of experiments uracil was also omitted from the growth medium. Cells extracts were prepared as described in ref.. Protein lysates were separated on 1-2. 5% SDS PAGE ties in and used in polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in phosphatebuffered saline containing 0. 05% Tween 20 for 30 min at room temperature. Membraneswere then incubated overnight at 4 Cwith primary antibodies directed against individual Bax, bovine PKC, yeast phosphoglycerate kinase, yeast Atg8p, GFP or yeast Por1p. Peroxidase coupled secondary antibodies were from Jackson ImmunoResearch Laboratories and membranes were incubated for 1 h at room temperature. Peroxidase activity was revealed by chemioluminescence. Mitochondria were isolated by differential centrifugation from zymolyase addressed Hedgehog inhibitor cells, as described previously. For carbonate and Triton X 100 removal, 1 mg of protein from isolated mitochondria was incubated in the presence of 0. 1 M Na2CO3 o-r Triton X 100 for 15 min and centrifuged for 15 min at 105,000?g. The presence of Bax h myc in the pellet and the supernatant was approved by Western blot. Assessment of cyt c material was measured by redox spectra of isolated mitochondria essentially as described previously.