Figure 4

HHT-enhanced TRAIL-induced necroptosis depends on activation of RIPK1/RIPK3 and can be blocked by necrostatin-1s (Nec-1s). (A, B) Cells were pretreated 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) or left untreated, and after one hour, 100 ng/ml of TRAIL were added. Cell lysates were prepared at the indicated time points after stimulation and RIPK1 (including its 42-kDa cleavage fragments) and RIPK3 were detected by Western blot. Detection of actin served as a loading control. (C) Cells were preincubated with 50 μM Nec-1s for one hour and 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 Nec-1s significantly reduced the level of necroptosis compared to cells not treated with Nec-1s, regardless of the presence of HHT or CHX. Asterisks indicate statistical significance (t-test; *, P < 0.05; **, P < 0.001; n.s., not significant). Co: Control, N: Necroptosis, A: Apoptosis.