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Transcription

1 doi: /nature11700 Figure 1: RIP3 as a potential Sirt2 interacting protein. Transfected Flag-tagged Sirt2 was immunoprecipitated from cells and eluted from the Sepharose beads using Flag peptide. The eluant was subjected to trypsin digestion and subsequently analyzed by mass spectroscopy and the human RefSeq database. Shown are proteins identified in the eluant that contained greater than two independent peptide fragments. 1

2 Figure 2: Absence of a detectable interaction between Sirt2 and RIP1. L929 cells were transfected with an empty vector (-) or with a vector encoding Flag-tagged Sirt2 (+) to assess potential interactions between Sirt2 and RIP1. As opposed to what was observed for RIP3 (see Figure 1a), no interaction between Sirt2 and RIP1 was detected. 2

3 Figure 3: Expression of Sirt2 in MEFs. Representative example demonstrating the level of Sirt2 expression in two independent isolates of either wild type (WT) or Sirt2 -/- MEFs. Actin is shown as a loading control. 3

4 Figure 4: Absence of RIP1-RIP3 complex formation in Sirt2 knockdown cells. Control L929 cells or L929 cells with stable shrna-mediated knockdown of Sirt2 were analyzed for their ability to form a RIP1-RIP3 complex following TNFα and z-vad-fmk (T/z) treatment. A RIP1- RIP3 complex is only observed in the control lentiviral-infected cells. 4

5 Figure 5: Assessment of endogenous RIP3 acetylation. Endogenous RIP3 acetylation was determined by immunoprecipitation of protein lysates using an acetyl-lysine specific antibody. Acetylation levels were assessed in the presence or absence of Sirt2 knockdown or in the presence or absence of TNFα and z-vad-fmk treatment (T/z). While RIP3 was acetylated under basal conditions, the level of acetylation was not altered by knockdown of Sirt2 or following the induction of necrosis. 5

6 Figure 6: Assessment of RIP1 acetylation. Reverse procedure for detecting RIP1 acetylation using immunoprecipitation of RIP1 followed by Western blotting using an acetyl-lysine antibody (AcK). These results are consistent with results shown in Figure 3 demonstrating that TNFα and z-vad-fmk treatment (T/z) catalyzes RIP1 deacetylation in a Sirt2-dependent fashion. 6

7 Figure 7: Assessment of endogenous RIP1 acetylation. Levels of acetylation for endogenous RIP1 were determined using L929 cells either in the presence or absence of stable Sirt2 knockdown or in the presence or absence of TNFα and z-vad-fmk treatment (T/z). As seen in Figure 3 for epitope-tagged RIP1, levels of endogenous RIP1 declined after necrotic stimulation in a Sirt2-dependent fashion. 7

8 Figure 8: Assessment of Sirt2 activity and NAD levels during necrosis. a, L929 cells were transfected with an empty control vector or with vector encoding for Flag-Sirt2. Samples were prepared with and without stimulation TNFα and z-vad-fmk treatment and protein lysates were subsequently immunoprecipitated with Flag antibody and assessed for Sirt2 specific activity. No detectable differences in Sirt2 activity was observed during necrosis. b, Relative NAD levels based on absorbance at 565 nm was measured in L929 cells under basal conditions and for the first three hours after the induction of necrosis. Both panels represent one representative experiment performed in triplicate. 8

9 Figure 9: Knockdown of RIP3 in L929 cells. Four separate clones (A-D) for stable RIP3 knockdown all showing high degree of knockdown efficiency. Clone B and D were used for the data shown in Figure 3 and these two clones were indistinguishable in their behavior regarding the regulation of RIP1 acetylation. 9

10 Figure 10: The p300 acetyltransferase regulates RIP1 acetylation. Levels of RIP1 acetylation in cells that are transfected as indicated with Flag-RIP1 and HA-tagged p300. As demonstrated, p300 expression dramatically increases basal RIP1 acetylation. 10

11 Figure 11: Lysine 530 of RIP1 is a target of the p300 acetyltransferase. a, Schematic of RIP1 demonstrating the RHIM domain (in blue) and the adjacent Lysine 530 (red). b, Lysine 530 of RIP1 is an in vitro target of acetylation by p300. A peptide corresponding to amino acids 529 to 546 of RIP1 was synthesized with a single lysine residue at position 530. In the absence of p300, only the non-acetylated peptide is seen. After p300 addition, a shift corresponding to the acetylated peptide is observed. These spectrums were obtained on a different mass spectroscopy unit then were used in Fig. 3f, hence the x-axis differs slightly. 11

12 Figure 12: Sirt1 expression does not alter RIP1 acetylation. Cells were transfected with an HAtagged form of RIP1 with or without an epitope-tagged Sirt1 and the level of RIP1 acetylation assessed by acetyl-lysine antibody-mediated immunoprecipitation. Expression of Sirt1 had no effects of RIP1 acetylation levels. See Figure 3g for comparative effect of Sirt2 expression on RIP

13 Figure 13: The role for Lysine 530 is necrosis-induced deacetylation. Unlike wild type RIP1 kinase, the RIP1 K530Q mutant is resistant to deacetylation after stimulation with TNFα and z- VAD-fmk (T/z). Experiments were performed in L929 cells. 13

14 Figure 14: Endogenous RIP1-RIP3 complex formation in myocardium. Complex formation was determined by immunopreciptiation of RIP1 and determination of co-precipitating RIP3. In the absence of AGK2, wild type (Sirt2 +/+ ) hearts demonstrate increased RIP1-RIP3 complex formation after ischemia/reperfusion (I/R) injury. This I/R-stimulated increase in RIP1-RIP3 complex formation is not observed in Sirt2 -/- hearts or in wild type hearts treated with AGK2. Shown is one of two similar experiments. 14

15 Figure 15: Sirt2 -/- mice have smaller infarcts after in vivo coronary artery occlusion. Wild type mice (n=3) and Sirt2 -/- mice (n=4) were subjected to 30 minutes of coronary occlusion followed by reperfusion. As is routinely done, the infarcted region of the myocardium was normalized to the area of myocardium at risk. Analysis was performed in a blinded fashion. Shown is the mean +/- SD, *p<

16 Figure 16: Effects of necrostain-1 on RIP1. a, Treatment with necrostatin-1 blocks the interaction of RIP1 and RIP3 following treatment with TNFα and z-vad-fmk (T/z). b, Necrostatin-1 treatment does not alter the ability of Sirt2 to deacetylate RIP1. RIP1 acetylation was determined by immunoprecipitation (IP) using an acetyl-lysine antibody (AcK) followed by Western blotting for the HA-RIP1 epitope. These experiments may be useful in understanding whether deacetylation is an initial or enforcing event for RIP1-RIP3 interaction, although caution is warranted given that both RIP1 and Sirt2 are overexpressed. 16