Figure 5

Inhibition of UCH-L1 protects from TNF-induced necroptosis. A. L929Ts cells were prestimulated for 3 h with 50 μM of the UCH-L1 inhibitor LDN57444 or left unstimulated before addition of 100 ng/ml TNF and 20 μM zVAD-fmk for 5 h. Subsequently, cell death was analyzed by PI staining and flow cytometry. Asterisks indicate statistical significance (t-test), ***p < 0.001. Micrographs additionally show the morphology of untreated L929Ts cells vs. necroptotic cells vs. cells protected by LDN57444. Scale bar: 100 μm. B. L929Ts cells were transfected with an siRNA that does not elicit an RNAi response (negative control, siCtr), with an siRNA specific for murine UCH-L1, and with an siRNA specific for murine RIPK3 (positive control for protection from necroptosis, siRIPK3) as described in “Methods.” 48 h after transfection, cells were treated with 100 ng/ml TNF and 20 μM zVAD-fmk for another 5 h before the decrease of intracellular ATP levels was determined as a marker for cell death. ATP levels are shown relative to controls that were not treated with TNF/zVAD. Asterisks indicate statistical significance (t-test), **p < 0.01, ***p < 0.001.