CYLD does not regulate RIP1 ubiquitination within the TNFR1 complex.

<p>(<b>A</b>) Recruitment of CYLD and A20 to the TNFR-1 complex. TNFR1 complex was purified from TNF, zVAD and CHX treated cells by immunoprecipitation (IP). The recruitment of RIP1, CYLD and A20 was assessed via Western Blot. Control IPs with isotype-matched IgG were included to show the specificity of binding to TNFR-1. (<b>B</b>) CYLD^+/+ and CYLD^-/- MEFs were treated with TNF for the indicated times. Recruitment of polyubiquitinated RIP1 to TNFR-1 was determined by Western Blot. (<b>C</b>) IκBα phosphorylation and degradation was normal in CYLD^-/- MEFs.</p

CYLD regulates the kinetics of RIP1-RIP3 necrosome assembly.

<p>(<b>A</b>) WT or CYLD^-/- MEFs were treated with TNF, LBW242 and zVAD-fmk for the indicated times. Recruitment of RIP1 to RIP3 was determined by Western Blot. (<b>B</b>) HT-29 cells expressing non-specific shRNA (NS) or CYLD shRNA were treated with TNF, LBW242 and zVAD-fmk. RIP3 complexes were immunoprecipitated and RIP1 association was determined by Western Blot. (<b>C</b>) HT-29 cells were treated with TNF, the SM LBW242 and zVAD-fmk. Caspase-8 complexes were immunoprecipitated, and recruitment of RIP1 and FADD was determined by Western blot. (D) HT-29 cells were treated with TNF, Smac mimetic and zVAD-fmk. FADD complexes were immunoprecipitated, and recruitment of RIP1 was determined by Western blot.</p

Impaired RIP1 and RIP3 phosphorylation in the absence of CYLD.

<p>(<b>A</b>) MEFs were treated with TNF, zVAD-fmk and the LBW242. Cell lysates were extracted by sequential detergent lysis in NP-40 and SDS as described in methods. RIP1 and RIP3 in each fraction were examined by Western blot. Note that phosphorylated RIP1 and RIP3 (p-RIP1 and p-RIP3) were exclusively detected in the SDS fractions. (<b>B</b>) HT-29 cells stably expressing non-specific (NS) shRNA or CYLD shRNA were treated with TNF, LBW242 and zVAD-fmk for the indicated times. Unmodified and phospho-RIP1 and phospho-RIP3 were analyzed by Western Blot.</p

CYLD regulates RIP1 ubiquitination in the necrosome.

<p>(<b>A</b>-<b>B</b>) Increased RIP1 ubiquitination in the absence of CYLD. (<b>A</b>) CYLD^+/+ and CYLD^-/- MEFs or (<b>B</b>) control and CYLD knock-down HT29 cells were treated with TNF, the Smac mimetic LBW242 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076841#B35" target="_blank">35</a>] and zVAD-fmk for the indicated times. Total lysis in 1% SDS was performed as described in the methods. The level of RIP1 ubiquitination was examined by Western Blot. NS: non-specific band. (<b>C</b>) Necrosome-associated RIP1 contains ubiquitin chains of different linkage types. Cells were treated with TNF and zVAD-fmk for 3 hours or left untreated. RIP3 immune complexes were denatured in urea, followed by immunoprecipitation with the indicated antibodies against ubiquitin. (<b>D</b>) Selective accumulation of poly-ubiquitinated proteins in RIP1 complexes in the NP-40 insoluble compartment. WT MEFs or CYLD^-/- MEFs were treated to undergo necrosis with TNF, zVAD-fmk, and CHX for the indicated times. Cells were lysed in NP-40 lysis buffer and insoluble material was solubilized with SDS. RIP1 was immunoprecipitated from both fractions followed by Western blot with the indicated antibodies.</p

Schematic diagram of the proposed mechanism of CYLD-mediated necrosis.

<p>Although CYLD is recruited to the TNFR-1 complex, it dose not deubiquitinate RIP1 within the this compartment. Upon transition to the cytosol, CYLD deubiquitinates RIP1 within the NP-40 insoluble fraction to actively promote necrosome phosphorylation and activation. In addition to regulating RIP1 ubiquitination in the necrosome, CYLD also controls downstream events of necrosis such as ROS production. </p

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.</p

Partial protection against TNF-induced necrosis by CYLD siRNAs.

<p>(<b>A</b>) FADD-deficient Jurkat cells were transiently transfected with the indicated siRNAs. Forty-eight hours post-transfection, the cells were treated with TNF to induce necrosis. The percentage cell death was determined by propidium iodide staining and flow cytometry. Western Blots on the right show the efficiency of gene silencing. Representative of three experiments. (<b>B</b>) HT-29 cells were stably transfected with either non-specific shRNA (control) or CYLD targeting shRNA (clones: G11-2 and G11-8). Cells were treated with TNF, LBW242 and zVAD-fmk. Cell viability was determined by MTS assay (Promega). Western blot shows that expression of CYLD was inhibited in the two selected clones, G11 #2 and G11 #8. Representative of three experiments. (<b>C</b>-<b>D</b>) L929 cells were transfected with the indicated siRNAs. Necrosis was induced with (<b>C</b>) TNF or (<b>D</b>) TNF and zVAD-fmk. Cell death was determined by MTS assay. The panel on the right shows the reduction in protein expression of the siRNA transfected cells. Representative of two experiments.</p

Induction of RIP1 associated kinase activity in the NP-40 insoluble compartment.

<p>(<b>A</b>) Schematic flowchart showing the procedures by which the different NP-40 soluble and insoluble fractions were obtained. (<b>B</b>) Accumulation of necrosis signaling adaptors in the NP-40 insoluble fractions. The indicated fractions were analyzed for levels of RIP1, RIP3 and CYLD in response to necrosis induction by TNF. β-actin was used as internal control. (<b>C</b>) The kinase activity of the necrosome is selectively activated in the NP-40 insoluble fractions. Cell lysates were subjected to differential centrifugation as described in (<b>A</b>). The RIP1 immune complexes were subjected to <i>in </i><i>vitro</i> kinase assays using histone H1 as substrate. RIP1 ubiquitination and recruitment of RIP3 and CYLD were determined by Western blot (lower panels). (<b>D</b>) Induction of kinase activity in the NP-40 insoluble fraction is partially dependent on RIP1 kinase activity. The P10 fractions were prepared and RIP1 immune complexes were isolated. Where indicated, 30 µM of Nec-1 was added to the <i>in </i><i>vitro</i> kinase assay. (<b>E</b>) Densitometry quantification of RIP1 ubiquitination in the different fractions in (<b>C</b>).</p

Knock-down of RIP1 expression in L929 cells did not result in caspase-dependent apoptosis.

<p>L929 cells were transfected with siRNA against (A) RIP1 or (B) TR4. Transfected cells were treated with zVAD-fmk, Nec-1 and TNF as indicated. Cell death was determined by MTS assay. (C) L929 cells transfected with the indicated siRNA or Jurkat 4E3 cells were treated with TNF. PARP-1 cleavage was determined by Western blot. (D) Cell lysates from (C) were tested for active caspase 3 as measured by cleavage of the substrate DEVD-AMC. Release of AMC was determined by increases in AMC fluorescence as described in materials and methods.</p

Nec-1 inhibits TNF-induced necrosis independent of RIP1 in L929 cells.

<p>(A–C) RIP1 is required for zVAD-fmk, but not TNF-induced necrosis in L929 cells. (A) L929 cells were transfected with the indicated siRNA oligonucleotides with HiPerfect (Qiagen). TR4 is the control siRNA against TRAIL-R4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023209#pone.0023209-Clancy1" target="_blank">[36]</a>. Cells were treated with (A) 50 µM zVAD-fmk or (B) 10 ng/ml mTNF and cell death was measured by MTS assay. (C) Western blot shows RIP1 and RIP3 expression in cells transfected with the indicated siRNAs. (D–E) RIP1 is required for TNF-induced necrosis in 3T3 fibroblasts. NIH 3T3 fibroblasts were transfected with the indicated siRNA. (D) Expression of RIP1, RIP3 and ß-actin in cells transfected with the indicated siRNA was determined by Western blot. (E) Necrosis was induced with 10 µM zVAD-fmk, 0.5 µg/ml cycloheximide and 10 ng/ml mTNF for 20 hours. Cell death was assessed as in (A).</p