Apaf*). For controls (-), the ‘‘empty’’ version of the plasmids were introduced. Found at: doi:10.1371/journal.pone.0007655.s009 (0.05 MB PDF) Figure S8 Validation of 3-component yeast-based Caspase assay reconstituting DISC. Yeast transformants were prepared to assess the performance of the Fas/FADD/Caspase-8/10 three-compo- nent systems. Two independent clones of each transformant were plated on leucine-deficient medium containing X-gal. Substrates included LETD-containing cleavable (S8) and LETG-containing non-cleavable (G8) transcription factors, while Caspase expression plasmids included WT pro-Caspases-8 (A) and -10 or a catalytic mutant of Caspase-10. (B) FADD was expressed at low levels from a DADH1 promoter. Note that the lacZ reporter gene was activated only when the combination of Fas, FADD, and either WT pro- Caspase-8 or -10 was co-expressed and only when a cleavable substrate (S8) was employed. (Note that the optimal tetrapeptide cleavage sequence is the same for Caspases-8 and -10). Transfor- mants: (A) Yeast transformants included EGY48-6op-LEU2/2op- lacZ/TEF-Fas-d-S8 (LETD)-TA/CYC1-Caspase-8-HA (S8,C8) EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-G8(LETG)-TA/CYC1- Caspase8-HA (G8,C8), without (-) or with Fas, and without (-) or with FADD, which were expressed from either ADH and DADH1 promoter, respectively, to achieve high expression of Fas and low expression of FADD. (B) Yeast transformants included: EGY48- 6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/CYC1-Caspase10- FLAG (S8,C10); EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-G8 (LETG)-TA/CYC1-Caspase10-FLAG (G8,C10); and EGY48- 6op-LEU2/2op-lacZ/GPD-Fas-d-S8 (LETD)-TA/CYC1-Caspa- se10(C358RA358)-FLAG (S8,C10(C358RA358)) each without (-) or with Fas, and without (-) or with FADD-expressing vector or the corresponding empty vectors. Found at: doi:10.1371/journal.pone.0007655.s010 (0.05 MB PDF) Figure S9 Flow chart for cDNA library screening using 3 component system - application to death receptor cloning-Example #1. The screening strategy is essentially the same as outlined in Figure 3. The yeast transformant EGY48-6op-LEU2/2op-lacZ/ TEF-Fas-d-S8(LETD)-TA/CYC1-Caspase8-HA/DADH1-FADD was transformed with a HepG2 cell cDNA library (oligo dT-primed, PADH promoter). Independent colonies (1.76106) appeared on growth plates within 48 hours. Cells were collected, pooled, and a portion (2.46107) was seeded on the leucine-deficient selection plates containing X-gal. Blue-colored colonies (n = 150) appeared within a week, 13 of which encoded DR4, (#XA514), and 15 clones encoded DR5 variant 2 (#ZA512), while the rest were false positives. Screening another cDNA library from HepG2 cells (random- primed, PADH promoter) yielded eight DR5 (#XD108) clones. Found at: doi:10.1371/journal.pone.0007655.s011 (0.05 MB PDF) Figure S10 Flow chart for cDNA library screening using 3 component system to clone death receptors - Example #2. The screening strategy is essentially the same as outlined in Figure 3. The yeast transformant EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d- S8(LETD)-TA/CYC1-Caspase8-HA/DADH1-FADD was trans- formed with a human liver cDNA library (random-primed, PADH promoter) and a HeLa cell cDNA library (random-primed, PADH promoter). Found at: doi:10.1371/journal.pone.0007655.s012 (0.04 MB PDF) Figure S11 Flow chart for cDNA library screening using 3- component system to to clone death receptors - Example #3. The Yeast-Based Protease Technlgys screening strategy is essentially the same as outlined in Figure 3. The yeast transformant EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d- S8(LETD)-TA/CYC1-Caspase8-HA/DADH1-FADD was trans- formed with a HEK293T cell cDNA library (oligo-dT-primed, PADH promoter) and a HEK293T cell cDNA library (random- primed, PADH promoter, HA-tagged). Found at: doi:10.1371/journal.pone.0007655.s013 (0.04 MB PDF) Figure S12 Schematic representation of 3-component system used for cloning adapter protein that links Fas to pro-Caspase-10. (A) The zymogen pro-Caspase-10 was expressed (from CYC1- promoter) with substrate containing the LETD-containing cleav- age element S8 (from plasmid p413-GPD-Fas-d-S8-TA/CYC1- Caspase-10-FLAG) in the yeast EGY48 expressing 6op-LEU2. Fas was also expressed without activating the Caspase-10 from plasmid p426-2op-lacZ/ADH-Fas. (B) Addition of FADD activates Caspase-10, releasing the transcription factor. Found at: doi:10.1371/journal.pone.0007655.s014 (0.04 MB PDF) Figure S13 Validation of adapter protein cloning system for Fas/FADD/Caspase-8/10: Reconstituted DISC. Yeast cell trans- formants were prepared to assess the performance of the Fas/ FADD/Caspase-8 or Fas/FADD/Caspase-10 three-component systems. Two independent clones of each transformant were plated on leucine-deficient medium containing X-gal and grown for 4 days. Substrates included LETD-containing cleavable (S8) and LETG-containing non-cleavable (G8) transcription factors, while Caspase expression plasmids included wild-type (WT) pro- Caspases-8 (A) and -10 or a catalytic mutant of Caspase-10 (C358RA358) (B). Note that the lacZ reporter gene was activated only when the combination of Fas, FADD, and either WT pro- Caspase-8 or -10 was co-expressed and only when a cleavable substrate (S8) was employed. Transformants: (A) Yeast cell transformants included: EGY48-6op-LEU2/2op-lacZ/TEF-Fas- d-S8 (LETD)-TA/CYC1-Caspase8-HA (S8,C8) and EGY48-6op- LEU2/2op-lacZ/TEF-Fas-d-G8 (LETG)-TA/CYC1-Caspase8- HA (G8,C8), with empty vector (-) or with plasmids encoding FADD (expressed from CYC1 promoter) or Fas (expressed from ADH promoter). (B) Yeast cell transformants included EGY48- 6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/CYC1-Caspase10- FLAG (S8,C10), EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-G8 (LETG)-TA/CYC1-Caspase10-FLAG (G8,C10), and EGY48- 6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/CYC1-Caspase10 (C358RA358)-FLAG (S8,C10(C358RA358)), each with empty vector (-) or with plasmids encoding FADD or Fas as above. Found at: doi:10.1371/journal.pone.0007655.s015 (0.05 MB PDF) Figure S14 Flow chart for cDNA library screening using 3 component system to clone adapters. Yeast transformant EGY48- 6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/ADH-Fas/CYC1- Caspase10-FLAG was transformed with a HEK293 cell cDNA library (random-primed, PADH promoter, HA-tagged). Independent colo- nies (,2.26106) appeared on growth plates within 48 hours. Cells were collected, pooled, and a portion (3.26107) was seeded on leucine-deficient selection plates containing X-gal. Blue-colored colonies (n = 200) appeared within a week, five of which encoded full-length Caspase-2 (#XA227), five encoded a fragment of Caspase- 2 (#ZA214), five encoded a fragment of Caspase-8 (#XA221), and 24 encoded full-length FADD (#XA212), while the rest were apparent false positives. Found at: doi:10.1371/journal.pone.0007655.s016 (0.03 MB PDF) PLoS ONE | www.plosone.org 16 October 2009 | Volume 4 | Issue 10 | e7655