Figure 6

Downregulation of RIPK3 and MLKL protects from TRAIL/zVAD/HHT-mediated necroptosis. Mz-ChA-1 and HT-29 cells were transfected using siPORT™ Amine with siRNAs specific for human RIPK1, RIPK3, or MLKL, or – as a negative control – with a non-target siRNA (NT) that does not elicit an RNAi response. 72 h after transfection, cells were treated with 50 μM zVAD in combination with 0.1 μM HHT (Mz-ChA-1) or 1 μM HHT (HT-29) for one hour before 100 ng/ml TRAIL were added. After 24 h, the decrease of intracellular ATP levels (A) and release of LDH (B) was determined as a marker for cell viability or cytotoxicity, respectively. ATP and LDH levels are shown relative to controls that were not treated with TRAIL/zVAD/HHT. ATP measurements are shown as means of five independent experiments with five parallel determinations each. In each LDH measurement, one out of two representative experiments with five parallel determinations each is shown. Error bars indicate the corresponding SDs. Asterisks indicate statistical significance (t-test; *, P < 0.05; **, P < 0.001). Control Western blots of untreated transfected cells were performed to verify the downregulation of endogenous human RIPK1, RIPK3 and MLKL. Detection of actin served as loading control.