Figure S9 Expression of V5-DC20-TRB3 did not affect CASP3/7 activation and apoptosis. (A) HeLa cells were transfected with the V5-DC20-TRB3 expression plasmid. Twenty- four hours after, cells were treated with TNFa/CHX for 4 hr. CASP3/7 activity was measured in V5-DC20-TRB3 expressing HeLa cells as described in the legend of Figure 2C. Error bars indicate mean 6SD of three independent experiments. (B) Twenty-four hours after transfection with the V5-DC20-TRB3 expression plasmid or control vector, HeLa cells were treated with TNFa/CHX for 4 hr. The resulting dead cells were counted by trypan blue staining. Error bars indicate mean 6SD of three independent experiments. (PDF) Figure S10 The effect of CASP2 on TRB3 under ER stress condition. Twenty-four hours after transfection with the indicated V5-TRB3 expression plasmid or control vector, HeLa cells were treated with tunicamycin in the absence or presence of CASP2 inhibitor z-VDVAD-FMK (10 mM) (BioVision) for 24 hr. The cell lysates were subjected to immunoblot analysis using anti- V5 antibody (A). Alternatively, the resulting dead cells were counted by trypan blue staining (B). Error bars indicate mean 6SD of four independent experiments. *P,0.005, **P,0.001. (PDF) Figure S11 The effect of TRB3 on ER stress independent apoptosis induced by staurosporine. Twenty-four hours after transfection with the indicated V5-TRB3 expression plasmid References 1. Bowers AJ, Scully S, Boylan JF (2003) SKIP3, a novel Drosophila tribbles ortholog, is overexpressed in human tumors and is regulated by hypoxia. Oncogene 22: 2823–2835. 2. Du K, Herzig S, Kulkarni RN, Montminy M (2003) TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver. Science 300: 1574–1577. 3. Kato S, Du K (2007) TRB3 modulates C2C12 differentiation by interfering with Akt activation. Biochem Biophys Res Commun 353: 933–938. 4. Kiss-Toth E, Bagstaff SM, Sung HY, Jozsa V, Dempsey C, et al. (2004) Human tribbles, a protein family controlling mitogen-activated protein kinase cascades. J Biol Chem 279: 42703–42708. 5. Ord D, Ord T (2003) Mouse NIPK interacts with ATF4 and affects its transcriptional activity. Exp Cell Res 286: 308–320. 6. Ohoka N, Yoshii S, Hattori T, Onozaki K, Hayashi H (2005) TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death. EMBO J 24: 1243–1255. 7. Bezy O, Vernochet C, Gesta S, Farmer SR, Kahn CR (2007) TRB3 blocks adipocyte differentiation through the inhibition of C/EBPbeta transcriptional activity. Mol Cell Biol 27: 6818–6831. 8. Takahashi Y, Ohoka N, Hayashi H, Sato R (2008) TRB3 suppresses adipocyte differentiation by negatively regulating PPARgamma transcriptional activity. J Lipid Res 49: 880–892. 9. Qi L, Heredia JE, Altarejos JY, Screaton R, Goebel N, et al. (2006) TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism. Science 312: 1763–1766. 10. Xu C, Bailly-Maitre B, Reed JC (2005) Endoplasmic reticulum stress: cell life and death decisions. J Clin Invest 115: 2656–2664. 11. Yoshida H (2007) ER stress and diseases. FEBS J 274: 630–658. 12. Woehlbier U, Hetz C (2011) Modulating stress responses by the UPRosome: A matter of life and death. Trends Biochem Sci 36: 329–337. 13. Jousse C, Deval C, Maurin AC, Parry L, Che´rasse Y, et al. (2007) TRB3 inhibits the transcriptional activation of stress-regulated genes by a negative feedback on the ATF4 pathway. J Biol Chem 282: 15851–15861. 14. Puthalakath H, O’Reilly LA, Gunn P, Lee L, Kelly PN, et al. (2007) ER stress triggers apoptosis by activating BH3-only protein Bim. Cell 129: 1337–1349. 15. Galehdar Z, Swan P, Fuerth B, Callaghan SM, Park DS, et al. (2010) Neuronal apoptosis induced by endoplasmic reticulum stress is regulated by ATF4-CHOP- mediated induction of the Bcl-2 homology 3-only member PUMA. J Neurosci 30: 16938–16948. 16. Zou CG, Cao XZ, Zhao YS, Gao SY, Li SD, et al. (2009) The molecular mechanism of endoplasmic reticulum stress-induced apoptosis in PC-12 neuronal cells: the protective effect of insulin-like growth factor I. Endocrinology 150: 277–285. 17. Schwarzer R, Dames S, Tondera D, Klippel A, Kaufmann J (2006) TRB3 is a PI 3-kinase dependent indicator for nutrient starvation. Cell Signal 18: 899–909. Nuclear Translocation of Procaspase-3 by TRB3 or control vector, HeLa cells were treated with staurosporine (STS) (50 nM) (Wako Pure Chemical Industries, Osaka, Japan) for 24 hr. The resulting dead cells were counted by trypan blue staining (A). Error bars indicate mean 6SD of four independent experiments. *P,0.005. Alternatively, the cell lysates were subjected to immunoblot analysis using anti-V5 antibody. Tunicamycin (5 mM) (B) or CASP9 inhibitor Ac-LEHD-CHO (10 mM) (Calbiochem) (C) was used for a comparison analysis of TRB3 cleavage. (PDF) Figure S12 A hypothetical model for the stress response mechanism of TRB3. (PDF) Movie S1 Nuclear distribution of proCASP3 is critical for the execution of apoptosis. (MOV) not Acknowledgments We thank Dr. K. Sakamaki for providing a mutant caspase-3 cDNA clone. Author Contributions Conceived and designed the experiments: KS ST YE TS. Performed the experiments: KS. Analyzed the data: KS ST TS. Contributed reagents/ materials/analysis tools: KS ST YE. Wrote the paper: KS TS. 18. Corcoran CA, Luo X, He Q, Jiang C, Huang Y, et al. (2005) Genotoxic and endoplasmic reticulum stresses differentially regulate TRB3 expression. Cancer Biol Ther 4: 1063–1067. 19. Inoue S, Browne G, Melino G, Cohen GM (2009) Ordering of caspases in cells undergoing apoptosis by the intrinsic pathway. Cell Death Differ 16: 1053–1061. 20. Wu¨rstle ML, Laussmann MA, Rehm M (2010) The caspase-8 dimerization/ dissociation balance is a highly potent regulator of caspase-8, -3, -6 signaling. J Biol Chem 285: 33209–33218. 21. Tadokoro D, Takahama S, Shimizu K, Hayashi S, Endo Y, et al. (2010) Characterization of a caspase-3-substrate kinome using an N- and C-terminally tagged protein kinase library produced by a cell-free system. Cell Death Dis 1: e89. 22. Ohoka N, Sakai S, Onozaki K, Nakanishi M, Hayashi H (2010) Anaphase- promoting complex/cyclosome-cdh1 mediates the ubiquitination and degrada- tion of TRB3. Biochem Biophys Res Commun 392: 289–294. 23. Mancini M, Nicholson DW, Roy S, Thornberry NA, Peterson EP, et al. (1998) The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling. J Cell Biol 140: 1485–1495. 24. Kamada S, Kikkawa U, Tsujimoto Y, Hunter T (2005) Nuclear translocation of caspase-3 is dependent on its proteolytic activation and recognition of a substrate- like protein(s). J Biol Chem 280: 857–860. 25. Takemoto K, Nagai T, Miyawaki A, Miura M (2003) Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects. J Cell Biol 160: 235–243. 26. Ord D, Meerits K, Ord T (2007) TRB3 protects cells against the growth inhibitory and cytotoxic effect of ATF4. Exp Cell Res 313: 3556–3567. 27. Wali VB, Bachawal SV, Sylvester PW (2009) Endoplasmic reticulum stress mediates gamma-tocotrienol-induced apoptosis in mammary tumor cells. Apoptosis 14: 1366–1377. 28. Humphrey RK, Newcomb CJ, Yu SM, Hao E, Yu D, et al. (2010) Mixed lineage kinase-3 stabilizes and functionally cooperates with TRIBBLES-3 to compromise mitochondrial integrity in cytokine-induced death of pancreatic beta cells. J Biol Chem 285: 22426–22436. 29. Szegezdi E, Logue SE, Gorman AM, Samali A (2006) Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep 7: 880–885. 30. Misiti F, Sampaolese B, Pezzotti M, Marini S, Coletta M, et al. (2005) Abeta(31– 35) peptide induce apoptosis in PC 12 cells: contrast with Abeta(25–35) peptide and examination of underlying mechanisms. Neurochem Int 46: 575–583. 31. Giri K, Ghosh U, Bhattacharyya NP, Basak S (2003) Caspase 8 mediated apoptotic cell death induced by beta-sheet forming polyalanine peptides. FEBS Lett 555: 380–384. 32. Cheung HH, Lynn Kelly N, Liston P, Korneluk RG (2006) Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: a role for the IAPs. Exp Cell Res 312: 2347–2357. PLoS ONE | www.plosone.org 12 August 2012 | Volume 7 | Issue 8 | e42721