1. NEMO and RIP1 Control Cell Fate in Response to Extensive DNA Damage via TNF-α Feedforward Signaling 
Sharon Biton, Avi Ashkenazi 
Cell 
Volume 145, Issue 1, Pages (April 2011)
DOI: /j.cell
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3. Figure 1
Extensive DNA Damage Induces Interaction of NEMO, RIP1, FADD, and Caspase-8
HeLa cells were pretreated with zVAD (20 μM) for 4 hr, followed by treatment with various doses of etoposide (Eto) for 20 hr (A) or 100 μM etoposide for the indicated period of time (B). Cell lysates were analyzed by immunoblot (IB) with specific antibodies to phosphorylated KAP1 or JNK, or to IκB.
(C–F) Cells were treated as above and analyzed by immunoprecipitation (IP) followed by IB as indicated.
(G–I) Cells were pretreated with DMSO or zVAD (20 μM) for 4 hr, followed by 100 μM etoposide for 20 hr, and analyzed by IP and IB as indicated. Iso: isotype-matched control for the IP Ab in the etoposide-treated cells. Asterisk: background band.
See also Figure S1.
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4. Figure 2 High-Dose Etoposide Induces ATM-Dependent C8 Activation
HeLa cells were treated with various etoposide doses for 20 hr and subjected to C8 IP followed by either IB analysis (A, top) or C8 enzymatic activity assay (A, bottom).
(B) Cells were treated with zVAD (20 μM) or DMSO for 4 hr, followed by 100 μM etoposide for 20 hr. Cell lysates were analyzed by IB as indicated.
(C) HeLa cells were transfected with control or NEMO siRNA for 48 hr, followed by treatment with 100 μM etoposide for 20 hr. Cell lysates were analyzed by NEMO IP, followed by C8 activity assay.
(D–G) HeLa cells were pretreated with the ATM-specific inhibitor Ku (ATMi) or vehicle plus zVAD for 4 hr, followed by treatment with 100 μM etoposide for 20 hr. Cell lysates were analyzed by IP and/or IB as indicated (D–F) or by C8 IP and activity assay (G).
(H–J) Cells were transfected with ATM or control siRNA for 48 hr, followed by treatment with 100 μM etoposide for 20 hr, and analyzed by IB as indicated (H) or by C8 IP, followed by IB (I) or by C8 activity assay (J). Pretreatment with zVAD was included before etoposide addition.
See also Figure S2.
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5. Figure 3
NEMO and RIP1 Operate Upstream of Cytokine Secretion and C8 Activation
(A–C) HeLa cells were transfected with control or NEMO siRNA for 48 hr, followed by treatment with 100 μM etoposide for 20 hr. Cell lysates were analyzed by IB as indicated (A), and supernatants were analyzed for IL-6 and IL-8 secretion by ELISA (B and C).
(D–F) Cells were transfected with NEMO or control siRNA for 48 hr, followed by etoposide as in (A)–(C). Cell lysates were analyzed by IP and IB as indicated (D and E), or by C8 IP and activity assay (F).
(G–J) Cells were transfected with RIP1 or control siRNA for 48 hr, followed by etoposide as in (A)–(C). Cell supernatants were analyzed for IL-8 secretion by ELISA (G), and cell lysates were analyzed by IP and IB (H and I), or by C8 IP and activity assay (J). Pretreatment with zVAD was included before etoposide addition.
Error bars in (B), (C), and (G) represent mean ± standard deviation (SD) of duplicates. See also Figure S3.
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6. Figure 4
Role of the IKK and JNK Pathways in the Response to High-Dose Etoposide
(A–E) HeLa cells were pretreated with the IKK-specific inhibitor BMS (IKKi, 5 μM) or vehicle for 4 hr, followed by treatment with 100 μM etoposide for 20 hr. Cell lysates were analyzed by IP and IB as indicated (A, C, and D), or by C8 IP and activity assay (E); cell supernatants were analyzed for IL-8 secretion by ELISA (B).
(F–J) Cells were prereated with the JNK-specific inhibitor SP (JNKi, 10 μM) or vehicle for 4 hr, followed by treatment with 100 μM etoposide for 20 hr. Cell lysates were analyzed by IP and IB as indicated (F, H, and I), or by C8 IP and activity assay (J); cell supernatants were analyzed for IL-8 secretion by ELISA (G). Pretreatment with zVAD was included before etoposide addition.
Error bars in (B) and (G) represent mean ± SD of duplicates. See also Figure S4.
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7. Figure 5
RIP1 Kinase Activity Is Required for IL-8 Secretion and C8 Activation
(A) HeLa cells were pretreated with the RIP1 kinase-specific inhibitor Nec1 (30 μM) or vehicle for 4 hr, followed by treatment with 100 μM etoposide for 20 hr. Cell lysates were analyzed by IB as indicated.
(B and C) Cells were pretreated with Nec1 (30 μM) or vehicle for 4 hr, followed by treatment with 100 μM etoposide for the indicated time, and cell lysates were analyzed by IB with Ab against phosphorylated p65 or IκB.
(D–G) Cells were treated as in (A) and cell supernatants were analyzed for IL-8 secretion by ELISA (D), or cell lysates were analyzed by IP and IB as indicated (E and F) or by C8 IP and activity assay (G).
(H) Cells were treated with 100 μM etoposide for 20 hr, and cell lysates were analyzed by RIP1 IP followed by IB using phospho-S/T Ab (clone 100G7E, see Experimental Procedures).
(I) Cells were treated with indicated etoposide doses for 2 hr or 22 hr, and cell lysates were analyzed as in (H).
(J and K) Cells were transfected with control, ATM (J), or NEMO siRNA (K) for 48 hr, followed by treatment with 100 μM etoposide for 20 hr, and analyzed as in (H).
(L and M) Cells were pretreated with IKKi (5 μM) versus vehicle (L) or Nec1 (30 μM) versus vehicle (M) for 4 hr, followed by treatment with 100 μM etoposide for 20 hr, and analyzed as in (H).
(N) Cells were transfected with the Δ300 RIP1 deletion construct or empty vector for 24 hr, followed by treatment with 100 μM etoposide for 20 hr, and analyzed as in (H).
(O–Q) Cells were treated with 100 μM etoposide for 20 hr and analyzed by NEMO (O) or FADD (P) IP followed by IB using phospho-S/T Ab. Vice versa, cell lysates were analyzed by IP using phospho-S/T Ab followed by IB with RIP1 and FADD Ab (Q). Pretreatment with zVAD was included before etoposide addition.
Error bars in (D) represent mean ± SD of duplicates. See also Figure S5 and Table S1.
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8. Figure 6
Autocrine TNF-α-TNFR1 Feedforward Signaling Mediates Induction of IL-8 Secretion and C8 Activation
(A and B) HeLa cells were treated with various doses of etoposide for 20 hr or with 100 μM etoposide for different periods of time. Cell supernatants were collected, concentrated 5-fold, and analyzed by TNF-α ELISA.
(C) HeLa cells were treated for 6 hr or 26 hr with 100 μM etoposide and the mRNA induction of TNF-α was measured by TaqMan Human NF-κB Pathway Array (see Experimental Procedures).
(D) Cells were pretreated with IKKi (5 μM) or vehicle for 4 hr, followed by treatment and analysis as in (A).
(E, G, and I) Cells were treated with 100 μM etoposide for the indicated time in the presence or absence of TNFR1-Fc (100 μg/ml). Cell lysates were analyzed by IB as indicated (E), or by RIP1 IP under denaturing conditions (G), or by NEMO IP (I), followed by IB as indicated.
(F) Cells were treated with 50 μM etoposide in the presence or absence of TNFR1-Fc for 20 hr, and cell lysates were analyzed by RIP1 IP and IB with anti-phospho-S/T Ab (clone 100G7E).
(H, J, and K) Cells were treated with 100 μM etoposide in the presence or absence of TNFR1-Fc for 20 hr, and cell supernatants were analyzed for IL-8 secretion by ELISA (H). Cell lysates were analyzed by IP and IB as indicated (J) or by C8 IP and activity assay (K).
(L–P) HeLa cells were transfected with control or TNFR1 siRNA for 48 hr and treated with 100 μM etoposide for 20 hr or as indicated. Cell lysates were analyzed by IB for KAP1 phosphorylation, IκB degradation, and JNK phosphorylation (L). Cell supernatants were analyzed by IL-8 ELISA (M). Cell lysates were analyzed by IP and IB as indicated (N and O) or by C8 IP and activity assay (P). Pretreatment with zVAD was included before etoposide addition.
Error bars in (C), (D), (H), and (M) represent mean ± SD of duplicates. See also Figure S6.
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9. Figure 7
A Model Depicting ATM Regulation of Cell Fate in Response to Low versus High Levels of DNA Damage
In response to a low level of DSBs, ATM activation induces ubiquitylation of NEMO/IKK-γ and its interaction with RIP1 and with IKK-α and -β, driving NF-κB activation. This early NF-κB activation phase is independent of RIP1 kinase activity and TNFR1 and promotes cell survival by inducing transcription of antiapoptotic genes such as cIAPs and cFLIP. Another key effect of NF-κB activation downstream of ATM is to set up an autocrine TNF-α-TNFR1 signaling loop. If the damage is resolved, this loop subsides. In contrast, if the level of DSBs is extensive, ATM activity is sustained and the feedforward signaling loop continues to operate. This accelerates further TNF-α production, supporting a second wave of NF-κB activity and driving TNFR1-mediated RIP1 phosphorylation. In turn, RIP1 kinase promotes JNK3-mediated induction of IL-8 production to alert other cells of the damage and recruits FADD to activate C8 and trigger programmed cell death. K63-linked polyubiquitylation of RIP1 by cIAPs, which occurs downstream of RIP1 kinase activation, promotes the second NF-κB activation phase; however, it can also counteract the recruitment of FADD and C8 to RIP1, providing an additional level of regulation.
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10. Figure S1
γH2AX Phosphorylation, NF-κB Stimulation, and C8 Activation in Response to DNA Damage, Related to Figure 1
(A) HeLa cells were treated with the indicated doses of etoposide (Eto) for 20 hr and phosphorylation of H2AX, BRCA1 and SMC1 was monitored by IB analysis.
(B) Cells were treated for up to 26 hr with 100 μM etoposide in the presence or absence of zVAD (20 μM), and γH2AX phosphorylation was measured by ELISA. Error bars represent mean ± SD of triplicates.
(C) Left panels: Cells were pretreated with 10 μM Ku (ATMi) or vehicle plus zVAD for 4 hr, followed by 20 hr treatment with 100 μM etoposide. Cell lysates were analyzed by NEMO IP followed by IB analysis of NEMO (long exposure in the upper panel shows ATM-dependent ubiquitylation of NEMO). KAP1 phosphorylation and I-κB degradation was also monitored by IB. Right panel: Cells were treated with 100 μM etoposide for 20 hr and NEMO IP was performed under denaturing conditions, followed by IB analysis with anti-NEMO or anti-ubiquitin antibody.
(D) Cells were treated for 20 hr with 100 μM etoposide. Cell lysates were analyzed by IP using RIP1 antibody followed by FADD IB.
(E and F) Cells were treated for 20 hr with 100 μM etoposide in the presence or absence of zVAD. (E) IB analysis of NEMO, RIP1, FADD, C8. (F) Cell lysates were analyzed by IP using FADD antibody followed by C8 activity assay.
(G) Cell lysates were analyzed by IP using RIP1 antibody followed by C8 activity assay.
(H and I) HeLa cells were transfected with control or C8 siRNA for 48 hr, followed by treatment with 100 μM etoposide for 20 hr. Cell lysates were analyzed by IP using C8 antibody followed by IB analysis for RIP1 and FADD (H) or by C8 activity assay (I). Iso indicates isotype-matched control for the IP antibody in the etoposide-treated cells.
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11. Figure S2
Recruitment of FADD and C8 to RIP1 Occurs in Response to Various DNA-Damaging Agents and in Different Cell Lines and Is Independent of p53, Related to Figure 2
(A and B) HeLa cells were treated with the following DNA damaging agents: Etoposide (Eto - 5, 50 μM), ionizing radiation (IR - 1, 10 Gy), Neocarzinostatin (NCS - 1, 5 μg/ml), Doxorubicin (Dox - 200, 500 ng/ml), Camptothecin (CPT - 10, 100 μg/ml) and incubated for 20 hr. Cell lysates were analyzed by C8 IP followed by IB analysis to detect association with FADD and RIP1 (A). KAP1 phosphorylation, IκB degradation, and JNK activation were monitored by IB analysis (B).
(C and D) HeLa, MDA231, MDA435 cells were treated for 20 hr with 100 μM etoposide in the presence of zVAD (20 μM). Cell lysates were analyzed by IP using C8 antibody followed by either IB analysis for association with RIP1 (C) or C8 activity assay (D).
(E–H) RKO cells were transfected with control or p53 siRNA for 48 hr, followed by treatment with 100 μM etoposide for 20 hr. Cell lysates were analyzed by IB as indicated (E), or by C8 IP followed by either IB analysis to detect association with FADD and RIP1 (F), or by C8 IP and activity assay (G), or cell supernatants were analyzed for IL-8 secretion by ELISA (H). Pretreatment with zVAD was included before etoposide addition.
Error bars in (H) represent mean ± SD of duplicates.
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12. Figure S3
Extensive DNA Damage Induces Production of IL-6 and IL-8 in ATM- and RIP1-Dependent Manner, Related to Figure 3
(A and B) HeLa cells were treated with 100 μM etoposide for the indicated time (A) or treated for 20 hr with the indicated doses of etoposide (B). Medium was collected, and secretion of IL-6 and IL-8 was analyzed by ELISA.
(C) HeLa cells were treated for 6 hr or 26 hr with 100 μM etoposide and the mRNA induction of IL-6, IL-8 was measured by TaqMan Human NFkB Pathway Array (see Experimental Procedures).
(D and E) HeLa cells were pretreated with 10 μM Ku (ATMi) or vehicle plus zVAD (20 μM) for 4 hr, followed by 20 hr treatment with 100 μM etoposide (D). Cells were transfected with control or ATM siRNA for 48 hr followed by treatment with 100 μM etoposide for 20 hr (E). Cell supernatants were analyzed for IL-8 secretion by ELISA.
(F) MDA231 and MDA435 cells were treated with 100 μM etoposide for 20 hr and cell supernatants were analyzed for IL-8 secretion by ELISA.
(G–I) MDA231 cells were transfected with control or RIP1 siRNA for 48 hr, followed by treatment with 100 μM etoposide for 20 hr. RIP1 levels, KAP1 phosphorylation, and JNK activation were monitored by IB (G). Cell supernatants were analyzed for IL-8 secretion by ELISA (H). Cell lysates were analyzed by IP using C8 antibody followed by caspase activity assay (I).
(J) MDA231 or (K) MDA435 cells were transfected with control, C8 or RIP1 siRNA for 24 hr and then treated with 100 μM etoposide for 48 hr or 96 hr respectively. Cell death was determined by FACS analysis of propidium iodide (PI) exclusion.
Error bars depict mean ± SD of duplicates in panels A–F and H and triplicates in panels J and K, and p values are based on t test analysis. Pretreatment with zVAD was included before etoposide addition except for panels J and K.
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13. Figure S4
The NF-κB Subunit RelA Supports Both Cytokine Secretion and C8 Activation while JNK3 Mediates the Former but Not the Latter Event, Related to Figure 4
(A–D) HeLa cells were transfected with control or RelA siRNA for 48 hr, followed by treatment with 100 μM etoposide for 20 hr. Cell lysates were analyzed by IB as indicated (A), and cell supernatants were analyzed for IL-8 secretion by ELISA (B). (C and D) Cell lysates were analyzed by IP and IB as indicated (C), or by C8 IP and activity assay (D).
(E) Cells were pretreated with BMS (IKKi) (5 μM) or vehicle for 4 hr, followed by treatment with 100 μM etoposide for 48 hr. Cell death was determined by FACS analysis of PI exclusion.
(F–I) HeLa cells were transfected with control or JNK3 siRNA for 48 hr, followed by treatment with 100 μM etoposide for 20 hr. Cell lysates were analyzed by IB as indicated (F), and cell supernatants were analyzed for IL-8 secretion by ELISA (G). (H and I) Cell lysates were analyzed by IP and IB as indicated (H), or by C8 IP and activity assay (I).
Error bars indicate mean ± SD of duplicates in panels B and G and triplicates in panel E, and the p values are based on t test analysis. Pretreatment with zVAD was included before etoposide addition except for panel E.
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14. Figure S5
Effect of the RIP1 Kinase Inhibitor Nec1 or a RIP1 Mutant Lacking the Kinase Domain on Etoposide-Induced Events, Related to Figure 5
(A) HeLa cells were treated for the indicated time with 100 μM etoposide and zVAD (20 μM) in the presence or absence of Nec1 (30 μM) and γH2AX phosphorylation was measured by ELISA.
(B and C) HeLa cells were treated for 6 hr or 26 hr in the presence or absence of Nec1 (30 μM) together with 100 μM etoposide and the mRNA induction of TNF-α and IL-8 was measured by TaqMan analysis (see Experimental Procedures).
(D–F) MDA231 cells were pretreated with Nec1 (100 μM) and zVAD (20 μM) for 4 hr followed by 20 hr treatment with 100 μM etoposide. Cell supernatants were analyzed for IL-8 secretion by ELISA (D). Cell lysates were analyzed by IP using C8 antibody followed by either caspase activity assay (E) or IB for RIP1 and FADD (F).
(G–J) HeLa cells were transfected with control, or PAK1 (G and H) or PKAcα (I and J) siRNA for 48 hr, followed by treatment with 100 μM etoposide for 20 hr and cell supernatants were analyzed for IL-8 secretion by ELISA (G and I). Cell lysates were analyzed by IP and IB as indicated (H and J).
(K and L) HeLa cells were transfected with control, PAK1(K) or PKAcα (L) siRNA for 48 hr, followed by treatment with 100 μM etoposide for 20 hr. Cell lysates were analyzed by RIP1 IP and IB using phospho-S/T antibody.
(M–P) Cells were transfected with a RIP1 mutant construct lacking the kinase domain (Δ300) or empty vector for 24 hr followed by treatment with 100 μM etoposide for 20 hr. (M) Cell supernatants were analyzed for IL-8 secretion by ELISA. Results represent means ± SD of the fold induction of IL-8 secretion from 3 independent experiments and the p value is based on t test. (N) Cell lysates were analyzed by IP using NEMO antibody followed by IB for RIP1 and FADD (N), or for the myc epitope tag (O) to confirm expression of Δ300. (P) Cell lysates were analyzed by C8 IP followed by caspase activity assay.
Error bars in (A)–(D), (G), and (I) represent mean ± SD of duplicates. Pretreatment with zVAD was included before etoposide addition.
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15. Figure S6
Extensive DNA Damage Induces K63-Linked Polyubiquitylation of RIP1, which Is Mediated by IAPs and Counteracts RIP1 Association with FADD and C8, Related to Figure 6
(A) HeLa cells were pretreated with either zVAD (20 μM) or DMSO for 4 hr, followed by 20 hr incubation with 100 μM etoposide. IP of RIP1 under denaturing conditions (see Experimental Procedures), followed by IB analysis with anti-ubiquitin antibody.
(B) Cells were treated as in (A). IP using K63 and K48 linkage-specific antibodies was performed as described previously (Newton et al., 2008), followed by IB analysis with anti-ubiquitin antibody.
(C and D) HeLa cells were pretreated with the IAP antagonist BV6 (2 μM) or vehicle for 4 hr, followed by treatment with 100 μM etoposide for 48 hr. IP of RIP1 under denaturing conditions was performed, followed by IB analysis with anti-ubiquitin antibody (C) or K63 linkage-specific antibody (D).
(E–G) Cells were treated as in (C). Cell lysates were analyzed by IB as indicated (note the inhibition of IκB degradation) (E). Cell lysates were analyzed by IP using C8 antibody followed by either IB analysis for association with RIP1 (F) or C8 activity assay (G).
(H) HeLa cells were pretreated with the IAP antagonist BV6 (2 μM) or vehicle for 4 hr, followed by treatment with 100 μM etoposide for 48 hr. Cell death was determined by FACS analysis of PI exclusion.
(I and J) Cells were transfected with control or cIAP2 siRNA for 48 hr and then treated with 100 μM etoposide for 20 hr. Cell lysates were analyzed by IP with C8 antibody followed by IB for RIP1 and FADD (I) or by caspase activity assay (J).
(K and L) Cells were treated with 50 μM etoposide in the presence of 10 μg/ml anti TNF-α for 20 hr. (K) Cell lysates were analyzed by IB as indicated (K) and cell supernatants were analyzed for IL-8 secretion by ELISA (L).
(M) Cells were pretreated with TNFR1-Fc for 4 hr and treated with 100 μM etoposide for 48 hr. Cell death was determined by FACS analysis of PI exclusion.
Error bars indicate mean ± SD of triplicates and the p value is based on t test analysis. Pretreatment with zVAD was included before etoposide addition except for panels H and M.
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