CA inhibits either the TRAIL or STS-induced necroptosis in presence of caspase inhibitor.

<p>U937 cells were treated either with STS (1 µM) or with TRAIL (50 ng/mL) in the presence or absence of zVD (5 µM) for 20 hrs. Nec (10 µM) or CA (10 µM) were added 1 hr before cell death was induced. (A-B) CA (10 µM) considerably inhibited the (A) TRAIL (n = 4) or (B) STS-triggered necroptosis (n = 3). Percentage of PI positive cells was determined. (C) Time course analysis of cells with depolarized mitochondria is shown after DiOC₆(3) staining of, unfixed cells for STS treatment combined with the indicated inhibitors (n = 2). Values are mean±SD. *, P<0.05, **, P<0.01 and ***, P<0.001 calculated by Student’s t-probe. (D) PS distribution in the plasma membrane is shown in representative dot plots of Annexin V-FITC and PI stained, unfixed cells analyzed by flow cytometry. The values indicate the percentage of cells in the marked regions (n = 2). (E) Morphological signs of apoptosis and necrosis are shown in representative fluorescent microscopic images (400x) of Hoechst/PI double stained U937 cells (n = 2). Scale bar on the first subfigure applies to all the figures in the panel. (F) Distribution of cells with various volumes of acidic compartments (endo-lysosomes) is shown in representative overlay histograms of AO stained cells analyzed by flow cytometry. (G) Column diagram of percentage of cells with high AO red fluorescence intensity (n = 2). (H) Distribution of cells with various mitochondrial transmembrane potential is shown in representative overlay histograms of DiOC₆(3) stained cells analyzed by flow cytometry. (I) Column diagram of percentage of cells with high DiOC₆(3) fluorescence intensity (n = 3). Values are mean±SD. *, P<0.05, **, P<0.01 and ***, P<0.001 calculated by Student’s t-probe.