After cleavage and removal of the DEVD peptides full read by caspase 3/7 activity, the fluorescence in each well was quantitated at an excitation wavelength of 485 20 nM and an emission wavelength of 535 25 nM and after correction based on blank control or the homogeneous caspase 3/7 reagent. Final fluorescent intensity was depicted as endpoint rela tive fluorescent Inhibitors,Modulators,Libraries unit, RFLU. Using similar experimental conditions Inhibitors,Modulators,Libraries but as an inde pendent study, the effect of LY294002 was also evaluated on growth and caspase 3/7 activity of cells treated with saposin C etoposide. After initial studies to find the optimal concentration, we used a non toxic tolerable dosage of 1. 5M for LY294002. In addition, we chose the most effective concen tration of etoposide and saposin C for this study.

Terminal deoxynucleotide transferase mediated nick end labeling Inhibitors,Modulators,Libraries Cells were cultured in multiwell chamber slides and treated with etoposide in the presence or absence of saposin C at 0. 1, 1, or 10 nM as indicated above. In situ determination of apoptosis by Terminal dUTP nick end labeling was performed using an ApopTag Per oxidase Inhibitors,Modulators,Libraries In Situ kit as recommended by the manufacturer. The ApopTag Kit detects single and double stranded DNA breaks asso ciated with apoptosis. Drug induced DNA damage is not identified by the TUNEL assay unless it is coupled with the apoptotic response. Briefly, at the end of the incubation period, cells were fixed in 1% paraformaldehyde in PBS, pH 7. 4 for 10 min at room temperature, washed with PBS twice, and permeabilized in pre cooled ethanol acetic acid for 5 min at 20 C.

After washing twice in PBS, 5 min each time, endogenous peroxidase activity in the cells was quenched in 3% H2O2 in PBS for 5 min at room temperature, incubated with terminal deoxynucleotidyl transferase and then with peroxidase con jugated Inhibitors,Modulators,Libraries anti digoxigenin antibody. Nuclear staining of the apoptotic cells was detected by 3,3 diaminobenzidine tetrahydrochloride dihydrate substrate, as recommended by the manufacturer. Cells were then counterstained in 0. 5% methyl green and slides were mounted under a glass coverslip in permount mounting medium. For control staining, the enzyme incubation step was deleted. Microscopic examination of cells was carried out using a phase contrast microscope. Cells were counted by choosing ten random fields and the molarity calculator percentages of apop totic cells were determined. Apoptosis was indicated by the presence of apoptotic bodies, exhibiting brightly labeled punctuated nuclei. Statistical analyses For cell survival and other quantitative data, a one way analysis of variance was employed to evaluate the influence of one variable on multiple independent groups. Bonferronis corrections were also applied when ever a significant group effect was observed.