Supplementary Figure 1. The chemical structure of compound A. Compound A has an amino terminal methyl alanine and compound B has an

Transcription

1 Supplemental Data IAP Antagonists Target ciap1 to Induce TNFα-Dependent Apoptosis James E. Vince, W. Wei-Lynn Wong, Nufail Khan, Rebecca Feltham, Diep Chau, Afsar U. Ahmed, Christopher A. Benetatos, Srinivas K. Chunduru, Stephen M. Condon, Mark McKinlay, Robert Brink, Martin Leverkus, Vinay Tergaonkar, Pascal Schneider, Bernard A. Callus, Frank Koentgen, David L. Vaux, and John Silke Supplementary Figure 1. The chemical structure of compound A. Compound A has an amino terminal methyl alanine and compound B has an amino terminal methyl glycine. Compound A has equivalent binding affinity to XIAP and ciap1 in the picomolar range and compound B is a weak binder with micromolar binding affinity to both IAPs. 1

2 Supplementary Figure 2. Compound A induces activation of NF-κB in primary and transformed cell lines. A) NF-κB activation was analyzed by flow cytometry of a polyclonal population of C2Cl2 myoblasts infected with an NF- B reporter lentivirus. Cells were treated with compound A or the inactive compound B overnight. Because there is no selection marker on the NF- B reporter lentivirus, cells that do not shift when treated with compound A represent uninfected cells and provide a convenient internal control. B) Primary MEF cells from the indicated genetic background were analyzed in an identical fashion to A). 2

3 Supplementary Figure 3. Schematic showing strategy used to investigate activation of NF-κB in conditional ciap1 MEFs. ciap1 conditional knock-in MEFs were generated from E15 conditional knock-in embryos using standard methods and then infected with the lentiviral NF-κB EGFP reporter. One clone was selected and then infected with cre-expressing lentivirus to knock out the ciap1 locus, and sub-clones selected with the results shown in Fig. 3G. In Fig. 3H a slightly different strategy was employed; a ciap1 knockout clone was infected with inducible ciap1 and a clone derived from a single cell was then infected with the NF-κB EGFP reporter. 3

4 Supplementary Figure 4. Compound A does not induce detectable production of TNFα in wild type, p65 or ciap1 knock-out MEFs. wt, ciap1 and p65 knock-out MEFs were treated with compound A in addition to Brefeldin A to inhibit secretion and promote accumulation of TNFα. Likewise TAPI was added to prevent cleavage and release of TNFα into the tissue culture media therefore also potentially increasing detectable TNFα in cell lysates. Neither of these treatments alone or in combination allowed detection of TNF in these cells leading us to conclude that compound A does not induce TNFα in MEFs. Similar experiments were performed in IAC insensitive human tumor cell lines with the same results. 4

5 Supplementary Figure 5. Blocking TNFa antibody can block IAP antagonist death. A) Compound A induced death of SKOV3 cells can be prevented by anti-tnfα antibody. SKOV3 were incubated with the indicated concentration of Compound A in the presence or absence of anti-tnfα blocking antibody. Cell viability was determined by MTT assay. Error bars are SEM of three independent experiments. B) Blocking TNFα antibody provides long term clonagenic survival to compound A treatment of Kym1 and OVCAR4 cell lines. Cells were treated as in Fig. 5A, except that after treatment, compound A was washed off and cells were replated and left to form colonies over 10 days. Colonies were stained with crystal violet. C) Natural IAP antagonist death can be blocked by anti-tnf blocking antibody. Inducible IAP antagonist lines were created as described in Figure 1. Blocking anti-tnf antibody (10µg/ml) was added simultaneously with the induction of the IAP antagonists and cell death measured 24hrs (Kym1) or 48hrs (SKOV3) later by PI staining and flow cytometry. Error bars are SEM of three independent experiments. 5

6 Supplementary Experimental Procedures Analysis of IAP antagonist binding to XIAP BIR3 domain. Binding constants (K d ) were measured using fluorescence polarization and the method of (Nikolovska-Coleska et al., 2004). Briefly, varying concentrations of compound A or compound B were mixed with 5 nm fluorescently labeled peptide (AbuRPF-K(5-Fam)-NH2) and 40nM of XIAP-BIR3 for 15 min at RT in 100μl of 0.1M Potassium Phosphate buffer, ph 7.5 containing 100μg/ml bovine γ-globulin. Following incubation, the polarization values (mp) were measured on a Victor 2 V using a 485nm excitation filter and a 520nm emission filter. IC 50 values were determined from the plot using nonlinear least-squares analysis with using GraphPad Prism (San Diego, CA). The K d values of competitive inhibitors were calculated using the newly derived equation described based upon the measured IC 50 values, the K d value of the probe and XIAP BIR3 complex, and the concentrations of the protein and probe in the competition assay. Western blotting and Immunoprecipitations For immunoprecipitation of endogenous TNF-R1, cells were grown on 15 cm tissue culture plates and when confluent, compound A was added for min. Cells were subsequently harvested and resuspended in 800 µl of ice-cold DME. Fc-TNFα (1.6 µg) was added for 30 min on ice (or indicated times at 37 C), cells washed thoroughly in ice-cold PBS, and then lysed in DISC buffer (1% NP-40, 10% glycerol, 150 mm NaCl, 20 mm Tris ph 7.5, 2 mm EDTA, Roche complete protease inhibitor cocktail) on ice. Cell lysate was spun at 14,000g, 10 min and the soluble material pre-cleared with Sepharose 6B beads at 4 C for 1 hr. Fc-TNFα bound material was immuno-precipitated by adding 6

7 the pre-cleared lysate to EZI view protein A agarose (Sigma) for 1hr at 4 C. Samples were washed 4 times with DISC buffer and then eluted with 1% SDS at 95 C for 3 min. Samples were reduced (β-mercaptoethanol), separated on 4-20% Tris-HCl polyacrylamide gels (Bio-Rad), and transferred to nitrocellulose membranes for antibody detection. All membrane blocking steps and antibody dilutions were performed with 5% skim milk in TBS containing 0.1% Tween 20 (TTBS) or 20% Horse Serum in PBS, and washing steps performed with TTBS. Antibodies used in this study were anti-iκb (Cell Signaling Technology, cat. #9242), TNF-R1 (Santa Cruz, cat. #sc-8436), ciap1 (in house), ß-actin (Sigma, cat. #A1978), RIP (mouse: BD transduction, cat Human: BD Pharmingen, cat ), FLAG-M2 (Sigma, cat. F3165), p65 (Santa Cruz cat. sc-372), phospho Ser536 p65 (Cell Signaling Technology, cat. 3033), TRAIL- R3 (Alexis, cat. #ALX C100) and TetraHis (Qiagen). All secondary antibodies used were conjugated to HRP and detected using ECL (Amersham). 7