ZNF216 Is an A20-like and I (cid:1) B Kinase (cid:2) -Interacting Inhibitor of NF (cid:1) B Activation*

The transcription factor NF (cid:1) B plays important roles in immune regulation, inflammatory responses, and an-ti-apoptosis. Activation of NF (cid:1) B requires the activity of I (cid:1) B kinase, a kinase complex that contains two catalytic subunits, IKK (cid:3) and IKK (cid:4) , and a non-enzymatic regulatory subunit, IKK (cid:2) . To understand how NF (cid:1) B activation is regulated at the IKK (cid:2) level, we searched for IKK (cid:2) interacting proteins by the yeast two-hybrid system. This search identified ZNF216, a zinc finger protein with unknown biological functions. ZNF216 contains an A20-like zinc finger domain (ZnF-A20) at its N terminus and an AN1-like domain (ZnF-AN1) at its C terminus. Similar to A20, ZNF216 interacted with IKK (cid:2) , RIP, and TRAF6 in co-immunoprecipitation experiments. Domain mapping experiments indicated that the ZnF-A20 domain was responsible for interacting with IKK (cid:2) and RIP, whereas the ZnF-AN1 domain interacted with TRAF6. ZNF216 tein, an A20-like zinc finger domain at its N terminus and an AN1-like zinc finger domain at its C terminus. In to also with and A20. Overexpression of ZNF216 inhibits NF (cid:1) B activation triggered by TNF, and Toll-like receptor 4 and sensitizes cells to TNF-in-duced apoptosis. Our findings suggest that ZNF216 and A20 have redundant and distinct functions in regulating NF (cid:1) B activation and apoptosis. (cid:2) ) were transfected with 3 (cid:5) g of an expression plasmid for FLAG-tagged A20 together with 3 (cid:5) g of an expression plasmid for HA-tagged ZNF216. Cell lysates immunoprecipitated with anti-FLAG antibody ( ) or control IgG Western blot analysis performed with anti-HA and anti-FLAG antibodies. The experiments repeated three times, and similar results obtained.

NFB contains five members in mammals, Rel (c-Rel), RelA (p65), RelB, NFB1 (p50 and its precursor p105), and NFB2 (p52 and its precursor p100) (1)(2)(3)(4). The major cellular form of NFB is a heterodimer consisting of the DNA binding subunit p50 and the transactivator p65. Normally, NFB is retained in the cytoplasm through association with its inhibitor IB. Upon stimulation by various NFB activating signals, IB is phosphorylated and degraded through a ubiquitin-dependent process. This process frees NFB, which is then translocated into the nucleus to activate transcription of downstream genes (1)(2)(3)(4).
A number of molecules, such as A20 (16 -22), ZIN (23), SINK (24), ABIN1 (18), and ABIN2 (25), have been implicated in negative regulation of NFB activation pathways. A20 was originally identified as a tumor necrosis factor (TNF)-responsive gene in human endothelial cells (16). A20 contains seven novel zinc finger motifs with characteristics of CX 2-4 CX 11 CX 2 C at its C-terminal domain (16). It has been shown that A20 interacts with RIP, IKK␥, and TRAF6 (13,17,18). Overexpression of A20 inhibits NFB activation triggered by various stimuli, including TNF, IL-1, and lipopolysaccharide (16 -22, 26). Gene knock-out studies have demonstrated that A20 is a suppressor of inflammation through attenuating TNF-induced NFB activation (21). A20 also can inhibit TNF-induced apoptosis, and a recent report suggests that this is mediated through disrupting recruitment of the death domain containing adapter proteins TRADD and RIP to the TNF receptor 1 signaling complex (27).
In this report, we identified a novel IKK␥-interacting pro-tein, ZNF216. ZNF216 contains an A20-like zinc finger (ZnF-A20) domain at its N terminus and an AN1-like zinc finger (ZnF-AN1) domain at its C terminus. In addition to IKK␥, ZNF216 also interacts with RIP, TRAF6, and A20. Overexpression of ZNF216 inhibits NFB activation triggered by TNF, IL-1, and Toll-like receptor 4 and sensitizes cells to TNF-induced apoptosis. Our findings suggest that ZNF216 and A20 have redundant and distinct functions in regulating NFB activation and apoptosis.
Constructs-The NFB (Dr. Gary Johnson, University of Colorado Health Sciences Center) and IRF-1 (Dr. Uli Schindler, Tularik Inc.) luciferase reporter plasmids were provided by the indicated investigators. The mammalian expression plasmids for HA-or FLAG-tagged RIP, IKK␤, IKK␥, TRAF2, TRAF6, and p65 were previously described (23,24). The HA-or FLAG-tagged mammalian expression plasmids for ZNF216 and its mutants were constructed by PCR amplification of the corresponding cDNA fragments and subsequently cloning into a cytomegalovirus promoter-based vector containing an N-terminal HA or FLAG tag.
Yeast Two-hybrid Screening-The cDNA encoding full-length human IKK␥ was inserted in-frame into the Gal4 DNA binding domain vector pGBT9 (Clontech). The human leukocyte two-hybrid cDNA library was screened and the isolation of positive clones was performed by following the manufacturer's protocols.
Northern Blot Analysis-Human multiple tissue mRNA blots were purchased from Clontech. The blots were hybridized with 32 P-labeled ZNF216 cDNA in the rapid hybridization buffer (Clontech) under high stringency conditions.
Transfection and Reporter Gene Assays-Transfection of 293 cells was performed with the standard calcium phosphate precipitation method (28). 293 cells (ϳ1 ϫ 10 5 ) were seeded on 12-well (25 mm) dishes and were transfected the following day. Within the same experiment, each transfection was performed in triplicate, and where necessary, empty control plasmid was added to ensure that each transfection received the same amount of total DNA. To normalize for transfection efficiency, 0.25 g of PRL-RSV-SV40 plasmid was added to each transfection. Dual specific luciferase reporter assays were performed using a luciferase assay kit (Promega, Madison, WI) by following the manufacturer's protocol. Firefly luciferase activities were normalized on the basis of Renilla luciferase expression levels.
Co-immunoprecipitation and Western Blot Analysis-Transfected 293 cells from each 100-mm dish were lysed in 1 ml of lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton, 1 mM EDTA, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). For each immunoprecipitation, a 0.4-ml aliquot of lysate was incubated with 0.5 g of the indicated monoclonal antibody or control mouse IgG and 25 l of a 1:1 slurry of Gamma Bind G Plus-Sepharose (Amersham Biosciences) for at least 2 h. The Sepharose beads were washed three times with 1 ml of lysis buffer containing 500 mM NaCl. The precipitates were fractionated on SDS-PAGE, and subsequent Western blot analysis was performed as described (23,24).
Apoptosis Assays-␤-Galactosidase co-transfection assays for determination of cell death were performed as described previously (29,30). Briefly, 293 cells (ϳ1 ϫ 10 5 ) were seeded on 12-well dishes and transfected the following day by calcium phosphate precipitation with 0.1 g of pCMV-␤-galactosidase plasmid and the indicated testing plasmids. Within the same experiment, each transfection was performed in triplicate, and where necessary, empty control plasmid was added to ensure each transfection received the same amount of DNA. Approximately 14 h after transfection, the cells were treated with TNF (20 ng/ml) or cycloheximide (10 g/ml) or left untreated for 12 h. Cells were then stained with X-gal as described previously (29,30). The number of survived blue cells from five representative viewing fields was determined microscopically. Data shown are averages and standard deviations of one representative experiment in which each transfection had been performed in triplicate.

Identification of ZNF216 as an IKK␥-interacting Protein-To
identify IKK␥-interacting proteins, we used the yeast two-hybrid system to screen a human leukocyte cell cDNA library with full-length IKK␥ as bait. We screened a total of ϳ6 ϫ 10 6 independent clones and obtained 33 ␤-galactosidase positive clones. Thirteen of these clones encoded ZNF216, a zinc finger protein expressed from human chromosome 9q (31). The biological functions of ZNF216 are unknown.
Sequence analysis suggested that ZNF216 was a 213 amino acid protein and contained two zinc finger domains (Fig. 1A). One zinc finger domain, ZnF-A20, was localized at amino acids 11-35 at the N terminus. ZnF-A20 contains a pattern of CX 2-4 -CX 11 CX 2 C that is similar to those found in A20, a zinc finger protein inhibiting TNF-induced NFB activation and apoptosis (16 -22). Another zinc finger domain, ZnF-AN1, was localized at amino acids 154 -193 at the C terminus. ZnF-AN1 contains a pattern of CX 2 CX 9 -12 CX 1-2 CX 4 CX 2 HX 5 HXC that is similar to that of AN1, a ubiquitin-like protein in Xenopus laevis (32 1. Structure and expression of human ZNF216. A, structure of human ZNF216. ZNF216 contains a ZnF-A20 domain at its N terminus and an ZnF-AN1 domain at its C terminus. B, Northern blot analysis of ZNF216 mRNA expression. PBL, peripheral blood leukocyte. C, protein expression of ZNF216. Human rhabdomyosarcoma RD and mouse myoblast C2C12 cells were treated with TNF (20 ng/ml) for 24 h, and cell lysates were analyzed by Western blot with an anti-ZNF216 antibody.
FIG. 2. ZNF216 interacts with IKK␥, RIP, and TRAF6 but not TRAF2. A, ZNF216 and its ZnF-A20 domain interact with IKK␥. 293 cells (ϳ2 ϫ 10 6 ) were transfected with 3 g of an expression plasmid for HA-tagged IKK␥, together with 3 g of an expression plasmid for FLAG-tagged ZNF216 or its mutants, ZNF216-(1-153) or ZNF216-(36 -213). Cell lysates were immunoprecipitated with anti-FLAG antibody (␣F) or control IgG (C), and Western blot analysis was performed with anti-HA antibody. Expression of IKK␥ was confirmed by Western blot analysis of the lysate (L) with anti-HA antibody. Expression of ZNF216 and its mutants was comparable as indicated by Western blot analysis of the lysates with anti-FLAG antibody (data not shown). B, ectopically expressed ZNF216 interacts with endogenous IKK␥. 293 cells (ϳ1 ϫ 10 7 ) were transfected with 20 g of an expression plasmid for ZNF216 for 24 h. Cells were lysed, and the lysate was immunoprecipitated with anti-ZNF216 antibody or preimmune serum (C). The immunoprecipitate was analyzed by Western blot with anti-IKK␥ antibody. C, ZNF216 and its ZnF-A20 domain interact with RIP. The same experiments were performed as in A, except that HA-tagged IKK␥ was replaced with HA-tagged RIP. D, ectopically expressed ZNF216 interacts with endogenous RIP. 293 cells (ϳ1 ϫ 10 7 ) were transfected with 20 g of an expression plasmid for ZNF216 for 24 h. Cells were lysed, and the lysate was immunoprecipitated with anti-ZNF216 antibody or preimmune serum (C). The immunoprecipitate was analyzed by Western blot with anti-RIP antibody. E, ZNF216 interacts with TRAF6 but not TRAF2. 293 cells (ϳ2 ϫ 10 6 ) were transfected with 3 g of an expression plasmid for FLAG-tagged TRAF6 (left panel) or FLAG-tagged TRAF2 (right panel), together with 3 g of an expression plasmid for HA-tagged ZNF216. Cell lysates were immunoprecipitated with anti-FLAG antibody (␣F) or control IgG (C), and Western blot analysis was performed with anti-HA and anti-FLAG antibodies. Expression of the indicated plasmids was confirmed by Western blot analysis of the lysates (L) with anti-HA and anti-FLAG antibodies. F, TRAF6 interacts with the ZnF-AN1 domain of ZNF216. 293 cells (ϳ2 ϫ 10 6 ) were transfected with 3 g of an expression plasmid for FLAG-tagged TRAF6, together with 3 g of an expression plasmid for HA-tagged ZNF216-(1-153) or ZNF216-(36 -213). Cell lysates were immunoprecipitated with anti-FLAG antibody (␣F) or control IgG (C), and Western blot analysis was performed with anti-HA and anti-FLAG antibodies. Expression of the indicated plasmids was confirmed by Western blot analysis of the lysates (L) with anti-HA and anti-FLAG antibodies. The above experiments were repeated three times, and similar results were obtained. was highly expressed in skeletal muscle. It was weakly expressed or undetectable in the brain, heart, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, and peripheral blood leukocytes (Fig. 1B).
To determine whether ZNF216 was expressed in mammalian cells at the protein level, we raised a rabbit polyclonal antiserum against recombinant full-length ZNF216. Western blot analysis suggested that ZNF216 was expressed as an ϳ33-kDa protein in both human rhabdomyosarcoma RD cells and mouse myoblast C2C12 cells (Fig. 1C).
ZNF216 Interacted with IKK␥, RIP, and TRAF6 in Mammalian Cells-To determine whether ZNF216 interacts with IKK␥ in mammalian cells, we transfected 293 cells with expression plasmids for FLAG-tagged ZNF216 and HA-tagged IKK␥ and IKK␤ and performed co-immunoprecipitation experiments. These experiments indicated that full-length ZNF216 interacted specifically with IKK␥ ( Fig. 2A) but not IKK␤ (data not shown).
We further examined whether endogenous ZNF216 and IKK␥ interact in untransfected cells in the presence or absence of TNF. These experiments failed to detect their interaction (data not shown). One explanation is that our polyclonal anti-ZNF216 antibody is not potent enough for this type of experiment, which requires high quality antibodies. Then we examined whether ectopically expressed FLAG-tagged ZNF216 could interact with endogenous IKK␥. The results indicated that ectopically expressed ZNF216 could interact with endogenous IKK␥ in 293 cells (Fig. 2B) and that this interaction was not affected by TNF stimulation (data not shown). In these experiments, IKK␥ was shown as two bands around 48 kDa, which is consistent with previous reports (8,11).
To determine which domain is responsible for the interaction of ZNF216 with IKK␥, we made ZNF216 deletion mutants containing either ZnF-A20 (amino acids 1-153) or ZnF-AN1 (amino acids 36 -213) and performed co-immunoprecipitation experiments. The results indicated that ZnF-A20 but not ZnF-

FIG. 3. ZNF216 or its ZnF-A20-containing mutant inhibits RIP-and TRAF6-but not p65-mediated NFB activation. A, ZNF216
inhibits RIP-and TRAF6-mediated NFB activation. 293 cells (ϳ1 ϫ 10 5 ) were transfected with 0.2 g of NFB-luciferase (firefly) reporter plasmid, 0.2 g of pRL-TK Renilla luciferase reporter plasmid, 1.0 g of an expression plasmid for RIP (white bars) or TRAF6 (black bars), and the indicated amounts (g) of ZNF216 expression plasmid. Sixteen h after transfection, firefly luciferase activities were measured and normalized based on Renilla luciferase levels. B, ZNF216 does not inhibit p65-mediated NFB activation. Reporter gene assays were performed as in A. ZNF216 and p65 expression plasmids were transfected alone or together as indicated. C, ZNF216-(1-153) inhibits RIP-and TRAF6-mediated NFB activation. Reporter gene assays were performed as in A, except that ZNF216 was replaced with ZNF216-(1-153). D, ZNF216-(36 -213) does not inhibit RIP-and TRAF6-mediated NFB activation. 293 cells (ϳ1 ϫ 10 5 ) were transfected with 1 g of an expression plasmid for RIP or TRAF6 as indicated, and 1 g of empty control plasmid (white bars) or an expression plasmid for ZNF216 (black bars). Reporter gene assays were performed as described in A. AN1 was required for the interaction of ZNF216 with IKK␥ ( Fig. 2A).
It has been reported that A20 interacts with RIP in the TNF-R1 complex and TRAF6 in the Toll-interleukin-1 receptor signaling complexes. Because ZNF216 contains an A20-like zinc finger domain, we determined whether ZNF216 can also interact with RIP and TRAF6. To test this possibility, we transfected 293 cells with an expression plasmid for ZNF216 together with an expression plasmid for RIP, TRAF6, or TRAF2. Co-immunoprecipitation experiments indicated that ZNF216 interacted with RIP (Fig. 2C) and TRAF6 (Fig. 2E) but not TRAF2 (Fig. 2E). Furthermore, ectopically expressed ZNF216 interacted with endogenous RIP in 293 cells (Fig. 2D).
Domain mapping experiments further indicated that ZnF-A20 was required for the interaction of ZNF216 with RIP (Fig. 2C), whereas ZnF-AN1 was required for the interaction of ZNF216 with TRAF6 (Fig. 2F).
ZNF216 Inhibited RIP-and TRAF6-but Not p65-induced NFB Activation-Because ZNF216 interacts with IKK␥, RIP, and TRAF6, we determined whether ZNF216 was involved in the regulation of NFB activation mediated by these signaling components. To do this, we performed NFB luciferase reporter gene assays in 293 cells. The results indicated that ZNF216 did not activate NFB but could inhibit RIP-and TRAF6-mediated NFB activation in a dose-dependent manner (Fig. 3, A and B).
In these experiments, ZNF216 did not inhibit p65-mediated To test the effects of ZNF216 or ZNF216-(1-153) on TLR4, 1 g of an expression for TLR4 was added to the transfection, and the transfected cells were left untreated for 15 h. Luciferase assays were performed as described in Fig. 3. C, ZNF216-(36 -213) does not inhibit TNF-, IL1-, and TLR4-induced NFB activation. Similar experiments were performed as in A and B, except that 1 g of an expression plasmid for ZNF216-(36 -213) was used to replace ZNF216 or ZNF216- (1-153). D, ZNF216 does not inhibit IFN-␥-induced IRF-1 activation. 293 cells (ϳ1 ϫ 10 5 ) were transfected with 0.5 g of an IRF-1 luciferase plasmid, 0.2 g of pRL-TK Renilla luciferase plasmid, and the indicated amounts (g) of ZNF216. Fourteen h after transfection, cells were treated with IFN-␥ (100 ng/ml) (black bars) or left untreated (white bars) for 6 h before luciferase assays were performed. Data shown are averages and standard deviations of relative firefly luciferase activities normalized based on Renilla luciferase levels.
NFB activation (Fig. 3B). Reporter gene assays further indicated that ZnF-A20 but not ZnF-AN1 was required for inhibiting NFB activation mediated by RIP and TRAF6 (Fig. 3, C  and D).
The Two Zinc Finger Domains of ZNF216 Interacted with Each Other, and ZNF216 Can Form Homo-oligomers-In the course of purifying recombinant ZNF216 protein, we found that ZNF216 existed mostly as homodimers or homotrimers (Fig.  5A). To confirm that ZNF216 can be self-associated in mammalian cells, we transfected 293 cells with FLAG-tagged and HA-tagged ZNF216 and performed co-immunoprecipitation experiments. The result indicated that ZNF216 could interact with itself or form homo-oligomers in 293 cells (Fig. 5B). To test whether an internal interaction existed between ZnF-A20 and ZnF-AN1, we transfected 293 cells with FLAG-tagged ZNF216-(1-153) and HA-tagged ZNF216-(36 -213) and performed coimmunoprecipitation experiments. The result indicated that the two zinc finger domains of ZNF216 could interact with each other (Fig. 5C).
ZNF216 Interacted with A20 -Because ZNF216 contained an A20-like zinc finger domain and acted similarly with A20 in inhibiting TNF-, IL-1-, and TLR4-triggered NFB activation, we determined whether ZNF216 can physically interact with A20. We transfected 293 cells with FLAG-tagged A20 and HA-tagged ZNF216 and performed co-immunoprecipitation experiments. The result indicated that ZNF216 interacted with A20 (Fig. 5D). These data suggest that ZNF216 can form a heterocomplex with A20.
ZNF216 Sensitized Cells to TNF-induced Apoptosis-Under most circumstances, NFB promotes cell survival through induction of anti-apoptotic genes, whereas NFB inactivation sensitizes cells to apoptosis. In this context, A20 represents an unconventional molecule. Although A20 inhibits TNF-induced NFB activation, it also inhibits TNF-induced apoptosis. To determine a potential role of ZNF216 in regulating apoptosis, we transfected 293 cells with ZNF216 and examined its effect on TNF-induced apoptosis. The results indicated that ZNF216 could sensitize cells to TNF-induced apoptosis (Fig. 6). DISCUSSION Under most conditions, inducible NFB activation is rapidly attenuated (1)(2)(3)(4). Several molecules that inhibit NFB activation pathways have been identified, such as A20, ZIN, SINK, ABIN1, and ABIN2 (16 -25). In this report, we identified ZNF216 as an additional inhibitor of NFB activation triggered by various stimuli.
ZNF216 shares several similar features with A20. It contains an A20-like zinc finger domain. Like A20, ZNF216 interacts with IKK␥ and RIP, and this interaction is medi- FIG. 5. Inter-and intramolecular interactions of ZNF216 and its formation of a heterocomplex with A20. A, ZNF216 exists as a monomer or oligomer. ZNF216 protein was expressed in Escherichia coli and purified with an Ni 2ϩ column. The purified protein was analyzed by gel filtration. B, ZNF216 forms homo-oligomers in mammalian cells. 293 cells (ϳ2 ϫ 10 6 ) were transfected with 3 g of an expression plasmid for FLAG-tagged ZNF216 together with 3 g of an expression plasmid for HA-tagged ZNF216. Cell lysate was immunoprecipitated with anti-FLAG antibody (␣F) or control IgG (C), and Western blot analysis was performed with anti-HA antibody. C, the two zinc finger domains of ZNF216 interact with each other. 293 cells (ϳ2 ϫ 10 6 ) were transfected with 3 g of an expression plasmid for FLAG-tagged ZNF216-(1-153) together with 3 g of an expression plasmid for HA-tagged ZNF216-(36 -213). Cell lysate was immunoprecipiated with anti-FLAG antibody (␣F) or control IgG (C), and Western blot analysis was performed with anti-HA and anti-FLAG antibodies. D, ZNF216 interacts with A20 in 293 cells. 293 cells (ϳ2 ϫ 10 6 ) were transfected with 3 g of an expression plasmid for FLAG-tagged A20 together with 3 g of an expression plasmid for HA-tagged ZNF216. Cell lysates was immunoprecipitated with anti-FLAG antibody (␣F) or control IgG (C), and Western blot analysis was performed with anti-HA and anti-FLAG antibodies. The experiments were repeated three times, and similar results were obtained. ated through its ZnF-A20 domain. ZNF216 also interacts with TRAF6, and this interaction is mediated through its ZnF-AN1 and/or the intermediate domain. Interestingly, A20 interacts with TRAF6 through its N-terminal domain, but not its seven ZnF-A20 domains at the C terminus (17). ZNF216 inhibits NFB activation triggered by TNF, IL-1, and TLR4, suggesting that ZNF216, similar to A20, is an IKK␥interacting common inhibitor of various NFB activating pathways.
ZNF216 also has features distinct from A20. ZNF216 contains only one ZnF-A20 domain, whereas A20 contains seven ZnF-A20 domains. ZNF216 contains a ZnF-AN1 domain at its C terminus; the functional significance of this domain is not fully understood. A20 is induced by TNF at the transcription level, but ZNF216 is not induced by TNF at either protein level (Fig. 1C) or mRNA level (data not shown). Most noticeably, ZNF216 sensitizes cells to TNF-induced apoptosis, whereas A20 inhibits TNF-induced apoptosis (17)(18)(19)(20)(21)(22). There are two simple explanations for ZNF216-mediated sensitization of cells to TNF-induced apoptosis. First, ZNF216 inhibits NFB activation and subsequent transcription of anti-apoptotic genes, and this sensitizes cells to TNF-induced apoptosis. Second, ZNF216 may directly neutralize the anti-apoptotic effect of A20 and therefore sensitize cells to TNF-induced apoptosis. Our demonstration of a physical interaction between ZNF216 and A20 is consistent with the latter hypothesis.
The N-terminal ZnF-A20 and C-terminal ZnF-AN1 domains of ZNF216 can interact with each other. It is possible that under physiological conditions ZNF216 is inactive because of this internal association. Upon stimulation, the two domains may dissociate, exposing the ZnF-A20 domain to interact with other molecules, such as A20, IKK␥, RIP, and TRAF6. The result of these processes may be responsible for ZNF216-medi-ated inhibition of NFB activation and sensitization of cells to apoptosis.
The physiological significance of ZNF216 needs to be further investigated. Interestingly, ZNF216 is expressed mostly in skeletal muscles. Previous studies have suggested that NFB activation is critically involved in TNF-induced skeletal muscle wasting in cachexia (33). Our future experiments will examine whether ZNF216 is a physiological inhibitor of TNF-induced cachexia.
FIG. 6. ZNF216 sensitizes cells to TNF-induced apoptosis. 293 cells (ϳ1 ϫ 10 5 ) were transfected with 0.1 g of CMV-␤-gal vector and 2 g of the indicated plasmids. Fourteen h after transfection, cells were treated with TNF (20 ng/ml), TNF (20 ng/ml) plus cycloheximide (CHX, 10 g/ml), or left untreated for 24 h. Cells were then stained with X-gal, and survival blue cells were counted. Data shown are averages and standard deviations of survival blue cell numbers from three independent experiments (transfection was performed in triplicate in each experiment).