were transformed with plasmids encoding Caspase-3 (‘‘Positive’’ control) or the empty vector (‘‘Negative’’ control). Found at: doi:10.1371/journal.pone.0007655.s006 (0.04 MB PDF) Figure S5 Validation of yeast-based assays for effector Caspase activators - two- and three-component systems. Yeast EGY191 strain containing 2op-LEU2/2op-lacZ or EGY48 strain containing 6op- LEU2/2op-lacZ were employed for developing assays for activators of downstream effector Caspases (e.g. Caspases-3 and -7). Yeast transformants were plated on leucine-deficient medium containing X-gal. Yeast were transformed with plasmids encoding membrane tethered transcription factor substrate with either DEVD-containing cleavable (S3) or DEVG-containing non-cleavable (G3) linkers, and with plasmids encoding pro-Caspase-3 (C3) or pro-Caspase-7 (C7) or the corresponding empty vector (-). (Note that the optimal tetrapeptide sequence for both Caspase-3 and Caspase-7 has previously been reported to be DEVD). Yeast transformants were as follows: (A) S3 Substrate/No Caspase: EGY191-2op-LEU2/2op-lacZ/DTEF3-Fas- d-S3 (DEVD)-TA; S3 Substrate/Caspase-3: EGY191-2op-LEU2/ 2op-lacZ/DTEF3-Fas-d-S3/7(DEVD)-TA/DCYC2-HA-Caspase3; G3 Substrate/Caspase-3: EGY191-2op-LEU2/2op-lacZ/DTEF3- Fas-d-G3/7(DEVG)-TA/DCYC2-Caspase-3; and (B) S3 Substrate/ No Caspase: EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S3(DEVD)- TA; S3 Substrate/Caspase-7: EGY48-6op-LEU2/2op-lacZ/TEF- Fas-d-S3(DEVD)-TA/CYC1-Caspase-7; G3 Substrate/Caspase-7: EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-G3(DEVG)-TA/CYC1- Caspase-7. These yeast transformants were then subsequently transformed with plasmids expressing a small amount of Caspase-9 (driven from the CYC promoter) or plasmids expressing large amounts of wild-type (WT) or catalytically-defective (C287RA287) Caspase-9 (driven from the TEF promoter), with or without an active form (gain of function mutant not requiring cytochrome c for activation) of Apaf-1 (driven by TEF-promoter) or the correspond- ing empty vectors (-) (Haraguchi M, Torii S, Matsuzawa S, et al. J Exp Med 2000;191:1709-20). Note that the large amount of Caspase-9 (expressed from TEF promoter) activated the cleavable S3(DEVD) substrate when co-expressed with pro-Caspase-3 or pro- Caspase-7, but not in their absence, thus constituting a 2- component system. No lacZ reporter gene activity was detected when the non-cleavable substrate (DEVG) was employed (G3). In contrast, expressing a small amount of Caspase-9 (from the CYC promoter) or the catalytically-defective Caspase9 (C287RA287) did not activate Caspase-3 or Caspase-7. However, co-expressing active Apaf-1* with a small amount of pro-Caspase-9 activated the lacZ reporter gene in yeast expressing pro-Caspase-3 or -7 (but not in the absence of these downstream effector Caspases), thus constituting a 3-component system. Found at: doi:10.1371/journal.pone.0007655.s007 (0.06 MB PDF) Figure S6 Validation of yeast-based assays for initiator Caspase activators: two-component systems. Yeast EGY48 strain containing 6op-LEU2/2op-lacZ was employed for developing 2-component assays for activators of upstream initiator Caspases (e.g. Caspases-1, 2, 8, 9, 10). Two independent clones of yeast transformants were plated on leucine-deficient medium containing X-gal. Yeast were transformed with plasmids encoding various membrane tethered transcription factor substrates containing (A) WEHD (‘‘S1’’), (B) DEHD (‘‘S2’’), (C) LEHD (‘‘S9’’) or (D) LETD (‘‘S8’’) cleavable linkers or their corresponding non-cleavable glycine mutants (‘‘G1’’, ‘‘G2’’, ‘‘G9’’, ‘‘G8’’). The yeast were also transformed with plasmids encoding wild-type (WT) or catalytically inactive mutants of proforms various initiator Caspases expressed from relatively weak promoters (e.g., CYC1; DTEF3), including (A) pro-Caspase-1, (B) Yeast-Based Protease Technlgys pro-Caspase-2, (C) pro-Caspase-9, and (D) pro-Caspase-10. (Note that the optimal betrapeptide sequences are the same for Caspase-8 and 10). These yeast were transformed with empty vectors (-) or plasmids encoding upstream activators of the Caspases, including (A) Asc, (B) RAIDD, (C) Apaf-1*, and (D) FADD, expressed from strong promoters (either GPD or TEF). Note that the lacZ reporter gene was activated only when the combination of an initiator Caspase and upstream activator was co-expressed, along with a cleavable substrate. Transformants: The transformed yeast cell clones are: (A) EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S1(WEHD)-TA/DTEF3- Caspase1-FLAG (S1,C1(WT)), EGY48-6op-LEU2/2op-lacZ/TEF- Fas-d-G1(WEHG)-TA/DTEF3-Caspase1-FLAG (G1,C1(WT)), or EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S1(WEHD)-TA/DTEF3- Caspase1(C285RG285)-FLAG, (S1,C1(C285RG285)), were trans- formed with the plasmids encoding the activator Asc, or the empty vector (-); (B) EGY48-6op-LEU2/2op-lacZ/DTEF2-Fas-d- S2(DEHD)-TA/DGPD1-HA-Caspase2- FLAG(S2,C2(WT)), EGY48- 6op-LEU2/2op-lacZ/DTEF1-Fas-d- G2(DEHG)-TA/DGPD1-HA- Caspase2-FLAG (G2,C2(WT)), or EGY48-6op-LEU2/2op-lacZ/ DTEF2-Fas-d-S2(DEHD)-TA/DGPD1-HA-Caspase2(C320RA320)- FLAG (S2,C2(C320RA320)), were transformed with plasmids encoding the activator RAIDD, or the empty vector (-); (C) EGY48- 6op-LEU2/2op-lacZ/TEF-Fas-d-S9(LEHD)-TA/TEF-HA-Caspase9 (S9,C9(WT)), EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-G9(LEHG)- TA/TEF-HA-Caspase9 (G9,C9(WT)), or EGY48-6op-LEU2/2op- lacZ/TEF-Fas-d-S9(LEHD)-TA/TEF-HA-Caspase9(C287RA287) (S9,C9(C287RA287)), were transformed with plasmids encoding an active form of Apaf-1 (Apaf*), or the empty vector (-); (D) EGY48- 6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/ADH-Caspase10- FLAG (S8,C10(WT)), EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d- G8(LETG)-TA/ADH-Caspase10-FLAG (G8,C10(WT)), or EGY48- 6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/ADH-Caspase10 (C358RA358)-FLAG (C10(C358RA358)), were transformed with the plasmids encoding the activator FADD, or the empty vector (-). The large amount of FADD is enough to activate Caspases-10 by itself. Found at: doi:10.1371/journal.pone.0007655.s008 (0.06 MB PDF) Figure S7 Specificity of upstream activators of initiator Caspases - tested by 2-component systems. Yeast transformants were prepared and tested as described in Figures S5 and S6, except a matrix of plasmid combinations was prepared to evaluate the specificity of upstream activators. The substrates and Caspases tested are indicated across the top, while the activators are indicated along the side. Note that results obtained were as predicted, with (1) Apaf- 1* activating pro-Caspase-9, but not other initiator Caspases; (2) RAIDD activating pro-Caspase-2, but not other Caspases; (3) FADD activating pro-Caspases-8 and 10, but not other Caspases, and (4) Asc activating pro-Caspase-1 and 8. Note that while Asc contains a CARD that pairs with a complementary CARD in pro-Caspase-1 and would not be necessarily predicted to activate the DED- containing protease Caspase-8, it has previously been reported that Asc is an activator of Caspase-8 (Hasegawa M. et.al. J Biol Chem 280:15122-30 (2005); Masumoto, J. et. al., Biochem. Biophys. Res. Commun 303: 69-73 (2003). Transformants: Transformed yeast clones were as follows: S1,C1:EGY48-6op-LEU2/2op-lacZ/TEF- Fas-d-S1(WEHD)-TA/DTEF3-Caspase1-FLAG; S2,C2:EGY48- 6op-LEU2/2op-lacZ/DTEF2-Fas-d-S2(DEHD)-TA/DGPD1-HA- Caspase2-FLAG; S8,C8:EGY48-6op-LEU2/2op-lacZ/GPD-Fas- d-S8/10(LETD)-TA/CYC1-Caspase8-HA; S9,C9:EGY48-6op- LEU2/2op-lacZ/TEF-Fas-d-S9(LEHD)-TA/TEF-HA-Caspase-9; S8,C10:EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8/10(LETD)- TA/ADH-Caspase10- FLAG. These cells were transformed with the plasmids encoding the activators (Asc, RAIDD, FADD, and PLoS ONE | www.plosone.org 15 October 2009 | Volume 4 | Issue 10 | e7655