Figure 3

HtrA2/Omi mediates TNF-induced necroptosis. A. Cells were left without, or pretreated for 2 h with 25 (Jurkat I42) or 50 μM (L929Ts, HT-29) of Ucf-101. Subsequently, cells were further incubated for 5 (L929Ts), 16 (HT-29) or 6 h (Jurkat I42) without or with addition of 100 ng/ml TNF, 20 (L929Ts, HT-29) or 50 μM (Jurkat I42) zVAD-fmk and 5 μg/ml CHX (HT-29) before cell death was analyzed. ***p < 0.001. Micrographs show the morphology of untreated vs. necroptotic cells vs. L929Ts cells protected by Ucf-101. Scale bar: 100 μm. B. L929Ts cells were left untreated or treated with TNF/zVAD with or without Ucf-101 as in A with addition of 50 μM TPCK or not and analyzed as in A. C. Cells were transfected with siRNAs specific for murine (L929Ts) or human HtrA2/Omi (Jurkat I42), or a negative control siRNA (siCtr). After 48 or 72 h, cells were treated with 100 ng/ml TNF and 20 (L929Ts) or 50 μM (Jurkat I42) zVAD-fmk for another 5 (L929Ts) or 6 h (Jurkat I42) before the decrease of intracellular ATP levels was determined as a marker for cell death. Control Western blots of transfected but untreated cells were performed to verify downregulation of endogenous murine or human HtrA2/Omi. Detection of actin served as a loading control. D. Upper panel: wild-type (WT) and HtrA2/Omi-deficient MEF were stimulated with 100 ng/ml TNF, 20 μM zVAD-fmk and 1 μg/ml CHX for 16 h before cell death was determined. ***p < 0.001. Control Western blots show the presence or absence of murine HtrA2/Omi. Detection of actin served as a loading control. Lower panel: micrographs show the morphology of untreated and TNF/zVAD/CHX-treated WT vs. HtrA2/Omi-deficient MEF. Scale bar: 100 μm.