S1knockdown (Fig

S1knockdown (Fig. role of PUMA in necroptosis. Our results demonstrate that PUMA is activated in a RIP3/MLKL-dependent manner and promotes signal amplification in TNF-driven necroptosis in vitro and in vivo in a positive feedback loop. Results Is Transcriptionally Activated During RIP1/RIP3-Dependent Necroptosis. RIP1/RIP3-dependent necroptosis can be induced in HT29 colon cancer cells in response to inhibitor of apoptosis protein (IAP) inhibition by SMAC mimetics and caspase inhibition by caspase inhibitors (5). We treated HT29 cells with the SMAC mimetic LBW-242 (L) and the pan-caspase inhibitor z-VAD-fmk (z-VAD; Z) to induce necroptosis. Induction of necroptosis was analyzed by several methods (Fig. 1and and Fig. S1mRNA expression. (shRNA were treated and analyzed as in are expressed as mean SD. = 3. **< 0.01. The treatment with RIP1 inhibitor Nec-1 abolished PUMA induction in both HT29 cells and MEFs undergoing necroptosis, coinciding with restoration of cell viability and suppression of HMGB1 release (Fig. 1 and or by shRNA suppressed induction of PUMA and necroptosis by L+Z in HT29 cells (Fig. 1and Fig. S1null Jurkat cells (Fig. S1is transcriptionally activated during RIP1/RIP3-dependent necroptosis in different cell types. PUMA Induction Requires MLKL and Is Mediated by Autocrine TNF- and Enhanced NF-B Activity. We investigated the mechanism of PUMA induction during necroptosis. Execution of necroptosis is characterized by formation of the necrosome complex and activation of MLKL through its phosphorylation (8). PUMA induction by L+Z in HT29 cells was detectable shortly after the onset of RIP3-dependent MLKL phosphorylation (Fig. 1and Fig. S1knockdown (Fig. 2and Fig. S1suppressed PUMA induction and necroptosis in HT29 and SW1463 cells treated with L+Z (Fig. 2and Fig. S2knockout (KO) in MEFs also abrogated PUMA induction, but did not inhibit cell death induced by T+L+Z (Fig. S2promoter (Fig. 2promoter reporter via an NF-B binding site (Fig. S2siRNA were treated with L+Z. (siRNA were treated with L+Z as in mRNA expression at 24 h (promoter JNJ 303 in HT29 cells treated as in for 24 h. (secretion at indicated time points in HT29 cells treated as in and are expressed as mean SD. = 3. *< 0.05. It has been shown that NF-B can be activated by RIP1 in necroptosis signaling (20). We detected two phases of NF-B activation by p65 phosphorylation (S536) (Fig. 2and and mRNA and secretion were markedly increased at 12C18 h and were suppressed by MLKL knockdown or inhibition (Fig. 2and Fig. S2promoter by L+Z could be suppressed by inhibition of TNF, RIP1, MLKL, or NF-B (Fig. S2is directly activated by NF-B via autocrine TNF- at the early execution stage of necroptosis following JNJ 303 MLKL activation. PUMA Contributes to Necroptosis in RIP3-Expressing Cells with JNJ 303 Caspase Inhibition. We asked whether PUMA plays a functional role in necroptotic death. Knockdown of by shRNA or siRNA largely suppressed cell viability loss, ATP depletion, PI staining, and HMGB1 release in HT29, LoVo, and SW1463 cells treated with necroptotic stimuli (Fig. 3and Fig. S3KO by CRISPR/Cas9 showed JNJ 303 similar phenotypes as and shRNA were treated with L+Z. (for 24 h. Black arrowheads indicate mitochondria, and white arrowheads indicate plasma membranes. (Scale bars: 2 m.) (shRNA treated with L+Z. (KO MEFs were treated with 20 ng/mL TNF-, 2 M LBW242, and 10 M z-VAD (T+L+Z) and analyzed as in and are expressed as mean SD. = 3. > 0.05; *< JNJ 303 0.05; **< 0.01. The pan-kinase inhibitor staurosporine (STS), a widely used apoptosis inducer, can induce necroptosis under certain conditions (21). PUMA can be induced by STS and RGS5 contributes to STS-induced apoptosis (22). depletion suppressed STS-induced and RIP3/MLKL-dependent necroptosis in RIP3-expressing HT29 and LoVo cells with caspase inhibition (Fig. S3 null Jurkat cells (Fig. S3KO in MEFs suppressed the necroptosis induced by T+L+Z (Fig. 3and KO modestly reduced the necroptosis induced by relatively high doses of TNF- and z-VAD (T+Z) (24), but had little or no effect on that induced by bacterial lipopolysaccharides (LPS) or Poly I:C in MEFs and bone marrow-derived macrophages (BMDMs) (Fig. S4 and or KO (24). We then tested whether PUMA induction alone is sufficient to induce necroptosis. Infection of.