solutions were added by using the WellMate from Matrix Technologies Corp. Controls were included with each plate, corresponding to cells treated with DMSO (without compounds) and cells cultured with and without X-gal. The plates were sealed with breathable sealing film (from Axygen Scientific) to reduce evaporation and transferred to 30uC incubators. After 48 hours, the plates were brought to room temperature, the breathable film removed and replaced with the transparent polyester tape seal, and the plates were mixed, pelleted briefly and read using a Beckman DTX 880, recording absorbance at 620 nm. Yeast-based counter-screen assays. EGY48 yeast inducibly (GAL1 promoter) expressing the LexA/B42 transcription factor or containing the empty vector were used to detect compounds that directly inhibit the lacZ reporter gene or that alter b-galactosidase activity. The plasmids employed were p424-GAL1 and p424-GAL1- TA (transcriptional activator) with transformed cells selected on tryptophan-deficient plates. Yeast expressing Caspase-1 activated by expression of high levels of Asc (DTEF3-Caspase-1-FLAG/TEF- HA-Asc) were used to detect compounds that cross-react with hits from the Fas/FADD/Caspase-8 screen. The Asc/Caspase-1 yeast cells were cultured and assayed under identical conditions to those for the primary NLRP1 HTS assay (see above), using cells expressing 6op-LEU2/2op-lacZ reporter genes and TEF-Fas-d-S1-TA substrate. Supporting Information Supplemental Methods S1 Found at: doi:10.1371/journal.pone.0007655.s001 (0.03 PDF) MB Table S1 Determination of IC50 values for zVAD-fmk inhibition of Caspases in yeast. Yeast expressing various Caspases alone (at high levels) or in combination with upstream activators (at low levels) and cleavable substrates containing appropriate tetrapeptides reorganized by these proteases were used in 384 well b-galactosidase activity assays to assess inhibition by zVAD-fmk. The compound was titrated into assays at various concentrations and percentage inhibition was determined. IC50 values were determined, using PRIZM software for analysis. Found at: doi:10.1371/journal.pone.0007655.s002 (0.01 MB PDF) Figure S1 Substrate sequence- and Caspase activity-dependent cleavage of the S1, S2, and S9. Yeast transformants were plated on leucine-deficient medium containing X-gal. (A) The recipient yeast cell strains, EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S1(WEHD)- TA (S1), or EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-G1(WEHG)- TA (G1) were transformed with the plasmids encoding the active wild type Caspase1 (WT), the catalytically-defective Caspase1 (C285RG285), and the empty vector (-). (B) The recipient yeast cell strains, EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S2(DEHD)-TA (S2), or EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-G2(DEHG)-TA (G2) were transformed with the plasmids encoding the active wild type Caspase2 (WT), the catalytically-defective Caspase2 (C320RA320), and the empty vector (-). (C) The recipient yeast cell strain, EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S9(LEHD)-TA (S9), or EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-G9(LEHG)-TA (G9), were transformed with the plasmids encoding the active wild type Caspase9 (WT), the catalytically-defective Caspase9 (C287RA287), and the empty vector (-). Found at: doi:10.1371/journal.pone.0007655.s003 (0.05 MB PDF) Figure S2 Flow chart for cDNA library screening using a reporter gene strategy based on cleavable transcription factor. The Yeast-Based Protease Technlgys example here is S1 substrate. The yeast strain EGY48 containing 6op-LEU2 and 2op-lacZ reporter genes and Caspase-cleavable substrate (expressed from TEF-Fas-d-S1-TA) was transformed (left) with a HEK293 cell cDNA library (oligo-dT-primed, PGAL1 promoter). Independent colonies of 5.16105 clones appeared on growth plates in 48 hours. Cells were collected, pooled, and a portion (3.66106) was seeded on leucine-deficient selection plates containing X-gal. Blue-colored colonies (n = 42) appeared within a week, of which 6 corresponded to clone #ZB311 encoding full- length Caspase-4 (which is known to cleave WEHD tetrapeptide [Thornberry, N.A, et. al., J. Biol. Chem. 272, 17907–17911 (1997)]), while the rest were false positives. (Right) Another cDNA library screen (HEK293 random-primed, ADH promoter used to drive expression) resulted in 26106 clones, which were pooled and 2.16107 cells were screened on leucine-deficient, X-gal-containing plates, resulting in 10 blue colonies. Five clones (including #XE501) encoding a fragment of Casapse-1 (L89-G403) were isolated. The remaining five clones were apparent false positives. Found at: doi:10.1371/journal.pone.0007655.s004 (0.04 MB PDF) Figure S3 Flow chart for cDNA library screening using reporter gene strategy based on cleavable transcription factor. The example here is S3/S7 substrate. Yeast strain EGY48 containing 6op- LEU2 and 2op-lacZ reporter genes and the DEVD-containing cleavable transcription factor (expressed from TEF-Fas-d-S3-TA) was transformed with a HEK293 cell cDNA library (random- primed, PGAL1 promoter, HA-tagged) (left). Independent colo- nies (1.06106) appeared on growth plates within 48 hours. Cells were collected, pooled, and a portion (5.06106) was seeded on the leucine-deficient selection plates containing X-gal. Blue-colored colonies (n = 21) appeared within a week, of which 3 clones (including #ZB331) encoded full-length Caspase-3, while the rest were false positives. In a similar experiment (right), another HEK293 cDNA library (oligo-dT-primed, PADH promoter) (1.86106 initial colonies) was screened, resulting in 9 positive clones (including #X8312) encoding full-length Caspase-7. The remaining five clones were false positives. Both cloned Caspases are known to cleave DEVD tetrapeptide [Thornberry, N.A, et. al., J. Biol. Chem. 272, 17907–17911 (1997)]. Found at: doi:10.1371/journal.pone.0007655.s005 (0.04 MB PDF) Figure S4 Use of one-component yeast-based Caspase activity assay for cDNA library screening. (A) The plasmids containing library cDNAs from Figure S2 were recovered and re-transformed into yeast cells containing 6op-LEU2 and 2op-lacZ reporter genes with either cleavable (WEHD-containing) (expressed from TEF- Fas-d-S1-TA) or non-cleavable (WEHG-containing) transcription factor substrate (expressed from TEF-Fas-d-G1-TA) to confirm whether they cleave S1 specifically. As controls, yeast were transformed with a plasmid encoding Caspase-1 (‘‘Positive’’ control) or the empty vector (‘‘Negative’’ control). Assays were performed in duplicate, with cells grown on selection plates for 4 days. Note that the cDNA library clones supported lacZ reporter gene activation only when co-expressed with the S1 cleavable substrate. (B) The cDNA library plasmids from Figure S3 were recovered and used to re-transform the yeast strains containing either the same cleavable DEVD-containing or DEVG non- cleavable transcription factor, expressed from TEF-Fas-d-S3-TA (‘‘S3’’ substrate) and TEF-Fas-d-G3-TA (‘‘G3’’ substrate), respec- tively. Assays were performed in duplicate and cells grown on selection plates for 4 days. Note that the two cDNA library clones activated the lacZ reporter gene only when co-expressed with cleavable S3 (DEVD-containing) substrate. As controls, yeast cells PLoS ONE | www.plosone.org 14 October 2009 | Volume 4 | Issue 10 | e7655