Figure 5

HHT-enhanced TRAIL-induced necroptosis occurs via activation of the downstream signaling component MLKL. (A) Activation of MLKL was checked by Western blot of its unphosphorylated and phosphorylated forms during a time course of 24 h. Cells were prestimulated with 50 μM zVAD-fmk 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), followed by addition of 100 ng/ml of TRAIL after one hour. Cell lysates were prepared at the indicated time points after stimulation and loaded on a SDS gel (20 μg per lane). The unphosphorylated and phosphorylated forms of MLKL were detected by Western blot and detection of actin served as loading control. (B) Phosphorylation of MLKL was not detected in Mz-ChA-1 and HT-29 cells after induction of apoptosis with 100 ng/ml TRAIL for the indicated time points. 20 μg of protein was analyzed per lane, actin served as loading control and necroptotic samples were loaded for control. (C) Cells were preincubated with necrosulfonamide (NSA, specific inhibitor of MLKL) for one hour and afterwards stimulated as indicated in (A). After 24 h, loss of membrane integrity was measured as a marker for cell death by flow cytometric detection of PI-positive cells. Cell death in the corresponding controls is shown for comparison (white bars). Each bar represents the means of two independent experiments with three parallel determinations each, error bars indicate the corresponding SDs. The addition of NSA significantly reduced the level of necroptosis compared to cells not treated with NSA, regardless of the presence of HHT or CHX. Asterisks indicate statistical significance (t-test; **, P < 0.001). As part of the same experiment shown in Figure 4C, the same negative control values were used. For clarity of presentation, the data of Nec-1s- (Figure 4C) and NSA-stimulated cells are shown in two separate figures. Co: Control, N: Necroptosis, A: Apoptosis.