The amount of LDH released from cells exposed to the different doses Ponatinib TNKS2 of GPS at 1, 4 or 24 hours post exposure was directly com pared to the amount of enzyme from untreated cells and cells treated for the lengthiest part of the experiment with 1% Triton X buffer. The mean of measurements for the spontaneous LDH activity in the culture media due to the presence of serum was sub tracted from all experimental values. The measurements taken at 1 hour post exposure were not significantly different among all three GPS doses. The average percentage of cytotoxicity at that expo sure time was about 5. 8%. At 4 hours post expo sure, the percentages were markedly different, demonstrating a dose and time dependent increase in cytotoxicity. Exposure to 1 puff GPS resulted Inhibitors,Modulators,Libraries in cytotoxic death of approximately 7.

15 2. 42% of the cells. The per centages were more than three fold and quadra fold higher for cells treated with 3 and 5 puffs GPS, respectively. It has to be noted, that the Inhibitors,Modulators,Libraries high standard deviation of the 5 puff data at 4 hours expo sure did not allow for a statistically significant differentia tion between the 3 and 5 puffs cytotoxicities, as determined by one way ANOVA analysis although both 3 and 5 puff data were significantly differ ent than the data for 1 puff. At 24 hours post exposure, LDH release from cells revealed the same cyto toxicity pattern. Cells treated with 1 puff GPS reached 42. 46 7. 07% cytotoxicity, which was significantly lower than the percentages recorded for the cells treated with either 3 or 5 puffs.

As with 4 hours post exposure, at 24 hours the percentages of cytotoxicity of the cells treated with the higher doses were not Inhibitors,Modulators,Libraries significantly differ ent. FACS analysis of Annexin V PI stained cells To determine the mode of cell death upon GPS treatment, Inhibitors,Modulators,Libraries cells were stained with AnnexinV PI and analysed by flow cytometry, 2 h post exposure. As seen in Figure 2, the per centage of the Annexin Inhibitors,Modulators,Libraries V stained populations in the control group and all groups of GPS treated cells were not significantly different. A clear dose dependent increase of cells stained with both Annexin V and PI was observed. The higher doses, green fluorescence increased remarkably, reaching sellekchem 48. 27 3. 18% for cells treated with 3 puffs and 75. 90 3. 07% for cells exposed to 5 puffs. The results were more prominent at 4 h post exposure, especially for cells treated with the higher doses. m depolarization ascended to 81. 90 0. 40% for the cell sample treated with 3 puffs and to 90. 24 1. 33% for cells exposed to 5 puffs GPS. Cells exposed to 1 puff, at 4 hours post exposure did not exhibit such a dramatic increase in the percentage of the population with disrupted m when compared to the equivalent at 2 hours post exposure.