Figure S15 Examples of cDNA cloning results. The clones that activated the reporter genes were characterized by recovery of cDNA library plasmids and retransformation into yeast expressing Fas and pro-Caspase-10 (A), pro-Caspase-10 without Fas (B), or neither (C). Among the positive clones were a full-length Caspase- 2 (#XA227), a fragment of Caspase-2 (#ZA214, V130-L312), a fragment of Caspase-8 (#XA221, M1-K438), and full-length FADD (#XA212). Assays were performed in duplicate, growing cells for 4 days on plates. Found at: doi:10.1371/journal.pone.0007655.s017 (0.05 MB PDF) Figure S16 Optimization of signal:noise ratio in microtiter plates: cell density. EGY48 cells containing the 3-component Fas/ FADD/Caspase-8 system and Caspase-8-cleavable reporter were seeded at 2.356104/well (A), 26105/well (B), or 3.766105/well (C) in 384 well plates to compare cell densities. Cells were cultured at 30uC. The activity of b-galactosidase was measured after 72 hrs at various times after initiating cultures (mean+std dev; n = 3), for cells grown without (white circles) or with (black circles and black squares) X-gal and grown in the absence (circles) or presence (black squares) of 100 mM zVAD-fmk Caspase inhibitor. Found at: doi:10.1371/journal.pone.0007655.s018 (0.04 MB PDF) Figure S17 Optimization of signal:noise ratio in microtiter plates: time and X-gal concentration. (A) EGY48 cells containing the 3-component Fas/FADD/Caspase-8 system and Caspase-8- cleavable reporter were grown at 26105 cells/well in 384 well plates, comparing b-galactosidase activity at various times (mean 6 std dev; n = 3), for cells grown in the absence (black squares) or presence (circles) of X-gal and in the absence (circles) or presence (squares) of 100 mM zVAD-fmk Caspase inhibitor. (B) EGY48 cells containing the 3-component NLRP1DLRR/Asc/ Caspase-1 system and Caspase-1-cleavable reporter were grown at 26105 cells/well in 384 well plates, comparing X-gal concentra- tions. The activity of b-galactosidase was measured after 72 hrs culture (mean 6 std dev; n = 3) for cells grown in the absence (red circles) or presence (all others) X-gal. Various concentrations of X-gal were compared. Found at: doi:10.1371/journal.pone.0007655.s019 (0.04 MB PDF) Figure S18 Determination of Z’ scores for NLRP1(NALP1) and Fas(CD95) 3-component yeast-based HTS assays using caspase inhibitor zVAD-fmk. (A) Yeast strain EGY48 harboring the NLRP1/ ASC/Caspase-1 proteolytic network (B) or the Fas/FADD/Caspase- 8 proteolytic network were cultured at 26105 cells/well for 48 hours in SD media containing 400 mg/ml X-gal with DMSO solvent control (blue) or zVAD in DMSO (100 mM final concentration) (purple). The Z’ scores were determined by comparison of the DMSO-treated samples with the zVAD-treated samples. Found at: doi:10.1371/journal.pone.0007655.s020 (0.34 MB PDF) Figure S19 Experiments with ATG4B-expressing yeast. (A) Determination of cell density dependence. The production of b- galactosidase activity (y-axis) in 384 well plate format was compared for various densities of EGY48 cells (x-axis) harboring plasmids encoding ATG4B and the LC3-containing substrate and lacZ reporter genes containing either 2 (red, orange) or 8 (blue, green) LexA-operators. Medium consisted of 1% galactose/2%raffinos, with 100 mg/ml X-gal. Cells were cultured at 30uC for 48 hrs, then absorbance was measured at OD 620 nm. Data represent mean6std dev (n = 4). (B) The time-course of b-galactosidase activity generation was compared for EGY48 cells containing Yeast-Based Protease Technlgys plasmids encoding LEU2 and lacZ reporter with (2 vs 8) LexA operators. Cells were plated and culture as described above. Data represent mean 6 std dev (n = 4). Found at: doi:10.1371/journal.pone.0007655.s021 (0.02 MB PDF) Figure S20 Plasmids for co-expression of Caspases and substrate cleavable transcription factors in yeast. The plasmid p413 was used as the backbone for these constructions, containing CEN/ ARS centromeric origin for low-copy episomal replication in yeast (S. cerevisiae) and HIS3 gene for selection in his3 yeast strains. (A) The plasmid p413-TEF-Fas-d-S1-TA, where expression of the Fas-LexA/B42 membrane tethered transcription factor with WEHD linker (Caspase-1/4/5 cleavable) is driven by the TEF promoter. (B) The plasmid p413-TEF-Fas-d-S1-TA/DTEF3- Caspase-1-FLAG, contains two additional transcriptional units, where expression of the Fas-LexA/B42 membrane tethered transcription factor with WEHD linker (Caspase-1/4/5 cleavable) is driven by the TEF promoter and expression of pro-Caspase-1 with C-terminal FLAG tag is driven by an attenuated TEF3 promoter (DTEF3). (C) The plasmid p413-TEF-Fas-d-S8-TA/ CYC1-Caspase-8-HA contains two additional transcriptional units, where expression of the Fas-LexA/B42 membrane tethered transcription factor with LETD linker (Caspase-8/10 cleavable) is driven by the TEF promoter and expression of pro-Caspase-8 with C-terminal HA tag is driven by CYC1 promoter. (D) The plasmid p413-GDP-Fas-dS8-TA/CYC1-Caspase-10-FLAG, similarly con- tains two additional transcriptional units, where expression of the same Caspase-8/10 cleavable Fas-LexA/B42 substrate as above is driven by GDP promoter and where expression of pro-Caspase-8 with C-terminal FLAG tag is driven by CYC1 promoter. Transcriptional termination elements from the CYC1 and ADH genes were employed as illustrated. Found at: doi:10.1371/journal.pone.0007655.s022 (0.07 MB PDF) Figure S21 Plasmids for expression of upstream activators of Caspases and lacZ reporter gene in yeast. The plasmid p426 was used as the backbone for these constructions, containing 2m plasmid origin for high-copy episomal replication in yeast and URA3 gene for selection in ura3 yeast strains. (A) The plasmid p426-2op-lacZ contains a lacZ gene driven by a minimal promoter containing two tandem copies of binding sites for the LexA transcription factor, followed by ADH gene termination element. (B) Plasmid p426-2op-lacZ/CYC1-HA-Asc contains an additional transcriptional unit, where expression of human cDNA encoding N-terminal HA-tagged Asc protein is driven by a CYC1 promoter, and followed by ADH gene termination element. (C) In plasmid p426-2op-lacZ/DADH1-FADD, expression of a human FADD cDNA is driven by DADH1 promoter and followed by ADH termination element. (D) Plasmid p426-2op-lacZ/ADH-Fas con- tains a human Fas(CD95) cDNA driven by ADH promoter, and followed by CYC1 gene termination element. Found at: doi:10.1371/journal.pone.0007655.s023 (0.05 MB PDF) Figure S22 Yeast expression plasmids for functional screening of cDNA libraries and expression of upstream activators of Caspases. The plasmid p424 was used as the backbone for these constructions, containing 2m plasmid origin for high-copy episomal replication in yeast (S. cerevisiae) and TRP1 gene for selection in trp1 yeast strains. (A) The plasmid p426-GAL1-cDNAs contains human cDNAs directionally cloned downstream of a GAL1 promoter, followed by CYC1 gene termination element. (B) Plasmid p424-CYC1-HA-Asc contains a human cDNA encoding N-terminal HA-tagged Asc protein cloned downstream of a CYC1 PLoS ONE | www.plosone.org 17 October 2009 | Volume 4 | Issue 10 | e7655