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Cells were treated as indicated for 20 hrs. (A) Cytospins were stained with hematoxylin-eosin. Blue arrows show the apoptotic, green ones the necrotic cells (400x) (representative of n?=?2). Scale bar on the first subfigure applies to all the figures in the panel. (B) Western blot analysis of fragmented PARP-1 protein. Full length PARP-1 is 116 kDa, cleaved PARP-1 fragment is 89 kDa (representative of n?=?2). (C) TRAIL-induced caspase activity in U937 cells. U937 cells were exposed to 50 ng/mL hr-TRAIL (114?281 aa) in the presence or absence of zVD (5 ?M) for the indicated period of time. The ordinate shows the slope of the measured DEVDase activity curves of a representative experiment carried out in triplicates. (D) FACS analysis of treated cells. Plasma membrane integrity was analyzed after PI staining of cells. Inserted values on histograms show the percentage of the marked population (representative of n?=?7). (E-F) Nec and GA protected U937 cells from TRAIL+zVD-induced necroptosis. U937 cell were exposed to TRAIL (50 ng/mL) in the presence or absence of zVD (5 ?M) and (E) Nec (10 ?M) or (F) GA (1 ?M) for 20 hrs. PI stained cells were analyzed for membrane permeability. Percentages of PI positive cells were determined (n?=?7 for Nec and n?=?4 for GA). 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