RIP1-dependent necrosis occurs in CYLD^-/- MEFs.

<p>Wild type (CYLD^+/+) and CYLD^-/- MEFs were treated with (<b>A</b>) TNF and zVAD, (<b>B</b>) TNF, Smac mimetic and zVAD, or (<b>C</b>) TNF, cycloheximide (CHX) and zVAD-fmk for 12 hours. Cell death was determined by staining with propidium iodide (PI) and analyzed via flow cytometry. The inset shows loss of CYLD expression in CYLD^-/- MEFs using a C-terminal specific CYLD antibody. In (<b>D</b>), cells were treated with 10 ng/ml TNF, CHX and zVAD-fmk and cell death was measured at the indicated times following treatment. Representative of three experiments. (<b>E</b>) Nec-1 inhibited TNF-induced necrosis in CYLD^-/- MEFs. Representative of two experiments. (<b>F</b>) CYLD^+/+ and CYLD^-/- MEFs were treated with TNF and CHX for 12 hours. Cell death was determined by PI staining and flow cytometry. Representative of three experiments. (<b>G</b>) MEFs were treated with 10 ng/ml TNF and CHX for the indicated times. Representative of two experiments. (<b>H</b>-<b>J</b>) MEFs were treated with (<b>H</b>) TRAIL, (<b>I</b>) FasL or (<b>J</b>) staurosporine (STS) for 12 hours. Cell death was determined by PI exclusion and flow cytometry.