Figure 8

Consecutive induction of necroptosis and apoptosis eliminates cancer cells with higher efficiency. Cells were stimulated as indicated with 50 μM zVAD-fmk or not and with HHT (Mz-ChA-1: 0.1 μM; HT-29: 1 μM) or CHX (Mz-ChA-1: 7.12 μM; HT-29: 17.79 μM). After one hour, 100 ng/ml of TRAIL were added or not as indicated. After 24 h, loss of membrane integrity was measured as a marker for cell death by flow cytometric detection of PI-positive cells (1^st treatment). In a parallel experiment, after 24 h of treatment, the supernatant was discarded and adherent, viable cells (green columns) were washed with medium. After 24 h of reconstitution in medium, cells were treated again as indicated. After another 24 h, cells were measured as before via PI-staining (2^nd treatment). Each bar represents the means of two independent experiments with three parallel determinations each, error bars indicate the corresponding SDs. In the 1^st treatment, both HHT and CHX treatments were performed as part of the same experiment using the same controls. Therefore, the same control values are shown in the two respective panels. The same applies to the 2^nd treatment. Asterisks indicate statistical significance (t-test; *, P < 0.05; **, P < 0.001; n.s., not significant). Due to the high levels of cell death of Mz-ChA-1 cells after the first induction of necroptosis, less than 40,000 cells could be measured after the second induction (#). Co: Control, N: Necroptosis, A: Apoptosis.