1. Volume 30, Issue 6, Pages 689-700 (June 2008)
cIAP1 and cIAP2 Facilitate Cancer Cell Survival by Functioning as E3 Ligases that Promote RIP1 Ubiquitination 
Mathieu J.M. Bertrand, Snezana Milutinovic, Kathleen M. Dickson, Wai Chi Ho, Alain Boudreault, Jon Durkin, John W. Gillard, James B. Jaquith, Stephen J. Morris, Philip A. Barker 
Molecular Cell 
Volume 30, Issue 6, Pages (June 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
AEG40730 Is a Dimeric ATPF Mimetic that Activates Caspase-8 and Induces Cancer Cell Death
(A) Chemical structures of AEG40730 and AEG40599, together with affinities of these compunds for GST-BIR3 domains from XIAP, cIAP1, and cIAP2, were determined in vitro.
(B) MDA-MB-231, MDA-MB-231CD, and SKOV3 cells were exposed to 5 nM AEG40730 for 24 hr, and cytotoxicity was assessed by LDH assay; ∗∗∗p < 0.001, error bars represent SEM.
(C) Annexin V levels on MDA-MB-231 cells treated with 5 nM AEG40730; ∗p < 0.01, error bars represent SEM.
(D) MDA-MB-231, MDA-MB-231CD, and SKOV3 cells exposed to AEG40730 for 24 hr were immunoblotted as indicated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2
AEG40730 Exposure Activates a Proapoptotic TNF-α Autocrine Loop in a Subset of Cancer Cells
(A) rtPCR was performed using primer sets and cell types indicated.
(B) MDA-MB-231 and SKOV3 cells were exposed to AEG40730, etanercept, and TRAIL antibodies for 24 hr and then immunoblotted as indicated.
(C) MDA-MB-231 and SKOV3 cells were exposed to AEG40730 and etanercept for 18 hr, and cytotoxicity was assessed by LDH assay.
(D) TNF-α protein levels in media conditioned by cells for 48 hr.
(E) Cells were exposed to 5 nM AEG40730 for 24 hr in the presence of TNF-α (20 ng/ml) or TRAIL (0.5 ng/ml), and cytotoxicity was assessed by LDH assay. ∗p < 0.05, ∗∗∗p < 0.001; error bars represent SEM.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3
AEG40730 Treatment Induces Autoubiquitination and Proteasomal Degradation of cIAP1, cIAP2, and XIAP
(A and B) MDA-MB-231 cells were exposed to increasing concentrations of AEG40730 for 6 or 24 hr (A) or were exposed to 5 nM AEG40730 for periods from 5 to 360 min (B), then lysed and immunoblotted.
(C) XIAP wild-type or null littermate-derived MEFs were exposed to 20 nM AEG40730 for the periods indicated, then lysed and immunoblotted.
(D) MDA-MB-231 cells were incubated with AEG40730 for 30 or 60 min, and cIAP1 was immunoprecipitated and immunoblotted.
(E) MDA-MB-231 cells were exposed to 10 nM AEG40730 for 18 hr in the presence of epoxomycin or lactacystin, then lysed and immunoblotted.
(F) HEK293 cells were transfected with wild-type cIAP2 or cIAP2∗ (containing a RING finger mutation) and exposed to AEG40730 for 6 hr and immunoblotted. Lanes 7 and 8 are a 10-fold dilution of the protein shown in lanes 5 and 6.
(G) In vitro ubiquitination of IAPs in the absence or presence of AEG Arrows indicate nonubiquitinated, full-length IAPs.
(H) In vitro ubiquitination of cIAP1 with AEG40730 or AEG40599.
(I) MDA-MB-231 cells were exposed to compounds for 6 hr, then immunoblotted.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4
Constitutive and TNF-α-Induced RIP1 Ubiquitination Is Abolished by AEG40730 Exposure
(A) MDA-MB-231 cells were exposed to 5 nM AEG40730 for 6 hr and then treated with TNF-α (20 ng/ml) for 15 min, and RIP1 was immunoprecipitated and immunoblotted.
(B) MDA-MB-231 cells were exposed to AEG40730 or AEG40599 for 6 hr, and RIP1 was immunoprecipitated and immunoblotted.
(C) Indicated cells were exposed to 5 nM AEG40730 for 6 hr, and RIP1 was immunoprecipitated and immunoblotted as above.
(D) MDA-MB-231CD cells were exposed to AEG40730 in the absence and presence of TNF-α (20 ng/ml) or TRAIL (0.5 ng/ml) for 24 hr and then photographed using a phase contrast microscope (upper panel) or analyzed for cytotoxicity using MTT assays (lower panel); ∗∗∗p < 0.001; error bars represent SEM.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

6. Figure 5 cIAP1 and cIAP2 Facilitate K63 Ubiquitination of RIP1
(A and B) MDA-MB-231 cells were transfected with siRNAs targeting TRAF2 (T2), cIAP1 (C1), cIAP2 (C1), or cIAP1+cIAP2 for 42 hr or were treated with AEG40730 (5 nM) for 6 hr. In (B), cells were exposed to TNF-α for the last 10 min. Lysates and immunoprecipitates were immunoblotted as indicated.
(C) Wild-type and cIAP1−/− MEFs were treated with TNF-α, and RIP1 was immunoprecipitated.
(D) In vitro ubiquitination assays were performed on RIP1 using GST-TRAF2, GST-cIAP1, or GST-cIAP2, with Ubc13/Uev1a as the E2 component. Left panel shows RIP1 ubiquitination; right panel shows free polyubiquitin chain formation.
(E) In vitro ubiquitination assays were performed using GST-cIAP1 or GST-cIAP2 with Ubc13/Uev1a and either wild-type ubiquitin, K48only ubiquitin, or K63only ubiquitin.
(F) Cells were transfected with siRNAs targeting cIAP1 (C1), cIAP2 (C1), cIAP1+cIAP2 (C1+C2), or TRAF2 (T2) for 42 hr. Cells were left untreated or were exposed to TNF-α for the last 15 min, then immunoblotted.
(G) MDA-MB-231 cells were treated with AEG40730 for 345 min, then exposed to TNF-α for 15 min and immunoblotted as indicated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

7. Figure 6 RIP1-Binding Partners Change with AEG40730 Treatment
(A) MDA-MB-231 cells were treated with 5 nM AEG40730 (6 hr), with TNF-α (20 ng/ml, for 15 min), or with the two together (AEG40730 for 345 min, then TNF-α for last 15 min), and RIP1 was immunoprecipitated.
(B) MDA-MB-231 cells were treated with AEG40730 (5 nM for 6 hr), and caspase-8 was immunoprecipitated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

8. Figure 7
AEG40730 Induces Cell Death through a RIP1-Dependent Mechanism
(A) MDA-MB-231 cells were treated with 5 nM AEG40730 and 0.5 μM epoxomycin for 6 hr, then lysed, and RIP1 was immunoprecipitated.
(B) MDA-MB-231 cells were treated with 5 nM AEG40730, 0.5 μM epoxomycin, and 20 ng/ml TNF-α and then immunoblotted.
(C) MDA-MB-231 cells were transfected with siRNA targeting distinct RIP1 mRNA regions (designated RIP1 siRNA-1 and RIP1 siRNA-2) for 24 hr, then exposed to AEG40730 and/or TNF-α for an additional 24 hr; cytotoxicity was assessed using LDH assays. ∗∗∗p < 0.001, error bars represent SEM.
(D) MDA-MB-231 cells were transfected with RIP1 siRNA-1 and siRNA-2 and, 36 hr later, were exposed to 5 nM AEG40730 and 20 ng/ml TNF-α for an additional 18 hr, then immunoblotted.
(E) Wild-type Jurkat cells treated with AEG40730 and/or TNF-α were lysed, and RIP1 was immunoprecipitated.
(F and G) Wild-type and RIP1 null Jurkat cells were treated with AEG40730 and/or TNF-α, and cells were assessed for cytotoxicity using LDH assays (F) or immunoblotted (G). For (F), ∗p < 0.001; error bars represent SEM.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions