Supporting Information

Transcription

1 Supporting Information He et al /pnas SI Methods Cell Culture. Mouse J774A.1 and RAW macrophages were obtained from ATCC and were cultured in MEM supplemented with 10% FS (Sigma) and 100 units/m penicillin/streptomycin (Invitrogen). Primary macrophages were derived from bone-marrow cells and were cultured for 7 d in MM (30% 929-cell conditioned medium, 20% FS, and 50% RPMI-1640). 929-cellconditioned medium was prepared by growing 929 cells in MEM plus 10% FS for 7 to 10 d. The medium containing macrophage colony stimulating factor secreted by 929 cells was harvested and passed through a 0.22-μm filter. Plasmids and sirna Oligos. Mouse RIP3 cna was amplified using RT-PCR from total RNA of J774A.1 cells and then cloned into a modified pcna3.1(+) plasmid (Invitrogen), which contains 3XFlag at the N terminus. For RHIM mutant RIP3 (RIP3-AAAA), residues within the RHIM region were mutated to four alanine residues. The mouse RIP3 sirna oligo mrip3 (5 - CCCGACGATGTCTTCTGTCAA-3 ) was from Qiagen. The mouse RIP1 sirna oligo mrip1 (5 -CCACUAGUCUGACU- GAUGA-3 ) were purchased from harmacon. Transfection and Generation of Stable Cell ines. sirna oligos were transfected to J774A.1 or RAW264.7 cells using INTERFERin from Polyplus Transfection. To generate RIP3- and RHIM mutant- (RIP3-AAAA) expressing cells, linearized plasmid NA was transfected into J774A.1 cells by electroporation using an Amaxa Nucleofector. Forty-eight hours after transfection, the cells were cultured in complete medium containing 0.5 mg/m G418 (Calbiochem). After 3 wk, clones were tested for expression of the transgene. Western lot Analysis and Immunoprecipitation. Cell pellet was collected and resuspended in lysis buffer (20 mm Tris-HCl, ph 7.4, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1 mm Na 3 VO 4,25 mm β-glycerol-phosphate, 0.1 mm PMSF, a Roche complete protease inhibitor set, and a Sigma phosphatase inhibitor set). The resuspended cell pellet or tissue was vortexed for 10 s, incubated on ice for 20 min, and centrifuged at 20,000 g for 20 min. The supernatants were collected for Western blot analysis or immunoprecipitation. For anti-flag immunoprecipitation, cell lysates were incubated with anti-flag M2 agarose (Sigma) overnight at 4 C. The next day, beads were washed with lysis buffer, and the immunoprecipitates were eluted off the beads using a low ph elution buffer (Pierce). Acid elution was neutralized by adding a 1/20 volume of 1 M Tris-HCl, ph 9.5. ROS Measurements. riefly, cells were treated as described in Fig. 6 A and C, and Figs. S4 S6, and H2CFA (5 μm) was added 30 min before collecting cells. The stained cells were analyzed with a SRII flow cytometer. Macrophage % Survival S+Z poly(i:c)+z TAK-242(2µM) TAK-242(10µM) Fig. S1. TAK-242 specifically blocked Toll-like receptor-4 (TR4)-induced necrosis of macrophages. one marrow-derived macrophages isolated from wild-type mice were treated with MSO or TAK-242 at the indicated concentration 2 h before treatment with MSO (, control), Smac mimetic/z-va (S+Z), PS/z-VA(), or poly(i:c)/z-va [poly(i:c)+z] for an additional 20 h. Cell viability was determined by measuring ATP levels. ata were represented as mean ± S of duplicates. A Time(min) p-ik WT mutant Time(min) p-i WT mutant Fig. S2. NF-κ activation during TR 3/TR4-induced necrosis. (A and ) one marrow-derived macrophages isolated from wild-type and Toll/I-1 receptor domain-containing adapter inducing IFN-β() mutant mice were treated as indicated. Cell lysates were collected and subjected to Western blot analysis of phosphorylated Iκα and β-actin levels., poly(i:c). 1of5

2 A IP: IP: Input Time(min) Input S+Z 1h 3h 6h 1h 3h 6h Elution Elution Fig. S3. specifically forms a complex with RIP3 upon TR3/TR4 activation, but not TNFR activation. (A) J774A.1 cells stably expressing flag-tagged wildtype RIP3 were treated with MSO (control) (), poly(i:c) (), or poly(i:c)/z-va () for the indicated time period. Cell lysates were immunoprecipitated with Flag beads as described in Methods. The immunocomplex was eluted with acid. The levels of and endogenous in the immunocomplex were determined by Western blot analysis., poly(i:c). () J774A.1 cells stably expressing flag-tagged wild-type were treated as indicated. Cell lysates were immunoprecipitated with Flag beads. The immunocomplex was eluted with acid. The levels of and endogenous in the immunocomplex were determined by Western blot analysis. All experiments were repeated at least three times with similar results. WT- WT- WT-Z 0.6% 3.8% WT-S WT-S+Z WT- 2.5% 43.1% 2.3% WT- WT-poly(I:C) WT-poly(I:C)+Z 21.9% 0.6% 21.3% Fig. S4. Reactive oxygen species (ROS) accumulation correlates with necrosis of macrophages. one marrow-derived macrophages from wild-type mice were treated as indicated for 5 h. ROS production was measured in the presence of CM-H2CFA by flow cytometry. 2of5

3 RIP3-/-- RIP3-/-- RIP3-/--Z 2.1% 2.4% RIP3-/--S RIP3-/--S+Z RIP3-/-- 3.4% 6.2% 5.8% RIP3-/-- RIP3-/--poly(I:C) RIP3-/--poly(I:C)+Z 3.7% 1.3% 2.6% Fig. S5. ROS accumulation correlates with necrosis of macrophages. one marrow-derived macrophages from RIP3 / mice were treated as indicated for 5 h. ROS production was measured in the presence of CM-H2CFA by flow cytometry. 3of5

4 - - -Z 1.0% 2.4% -S -S+Z - 1.8% 37.7% 2.1% - -poly(i:c) -poly(i:c)+z 7.0% 0.9% 5.1% Fig. S6. ROS accumulation correlates with necrosis of macrophages. one marrow-derived macrophages from ps2/ps2 mice were treated as indicated for 5 h. ROS production was measured in the presence of CM-H2CFA by flow cytometry. 4of5

5 A Control RIP1 mrip1 Fig. S7. Necrostatin-1 blocks TR 3/TR4-induced necrosis. (A) RAW264.7 cells were transfected with control or RIP1 sirna oligos. Forty-eight hours posttransfection, cells were treated as indicated for an additional 24 h, and then cell viability was determined by measuring ATP levels. ata were represented as mean ± S of duplicates. Cell lysates were collected 48 h posttransfection, and 30-μg aliquots thereof were subjected to Western blot analysis of RIP1 and β-actin levels. z-va was used at 40 μm. () one marrow-derived macrophages from wild-type mice were treated with MSO or Necrostatin-1 (100 μm) 2 h before the treatment of MSO (control) (), Smac mimetic/z-va (S+Z), PS/z-VA (), or poly(i:c)/z-va [poly(i:c)+z] for an additional 20 h. Nec-1, Necrostatin-1. Cell viability was determined by measuring ATP levels. ata were represented as mean ± S of duplicates. All experiments were repeated at least three times with similar results. 5of5