replication (2m and CEN/ARS). To further control expression levels, we made several attenuated forms of the promoters by PCR- assisted deletional mutagenesis. Using a standard indicator gene, we determined the approximate relative strength of the promoters to be: DCYC4 , DCYC2 , CYC1 , DGPD2 , DGPD1 , DADH1 , ADH , DTEF3 , DTEF2 , DTEF1 , TEF. However, the relative locations of the promoters in complex plasmids somewhat affects their strength, especially when two or three genes are contained in one plasmid. In this regard, we sometimes co- expressed two or three genes within one plasmid by placing the genes flanked by the above promoters and inserting transcription termination elements between them, because the selectable marker genes available are limited (URA3, HIS3, TRP1, and LEU2). In addition, many of the expressed Caspases and upstream activators were cloned with N-terminal HA or C-terminal HA or FLAG epitope tags for convenience of detection of the protein products by immunoblotting. Examples of complex plasmids are (a) p413-TEF- Fas-d-S8-TA/CYC1-Caspase8-HA, which consists of CEN/ARS origin, HIS3 marker, Fas-d-S8-TA substrate driven by TEF promoter, and a C-terminally HA-tagged pro-Caspase-8 cDNA under control of the CYC1 promoter, as in Figure S20C, and (b) p426-2op-lacZ/DADH1-FADD, which consists of 2m origin, URA3 marker, lacZ gene under the control of two lexA operators, and a FADD gene with expression driven by a short form of the ADH promoter (DADH1), as in Figure S21C. Further details about plasmid constructions are provided as Supplemental Information. Construction of expression cDNA libraries. Oligo(dT)- primed or random heptamer-primed cDNA libraries were made in modified p424-GAL1, p424-GAL1-HA, p424-ADH, or p424- ADH-HA plasmids (carrying a TRP1 marker) using mRNAs derived from HEK 293 cells, HepG2 cells, HeLa cells, human liver, or human placenta, as in Supplemental Figure S22A. The mRNAs of HepG2 cells, HeLa cells, human liver, and human placenta were purchased from Ambion. The p424-GAL1 was modified to create 59XhoI and 39NotI sites downstream of a GAL1 promoter for insertion of the cDNAs. The p424-GAL1-HA has a HA-tag between the GAL1 promoter and the 59XhoI site. The p424-ADH and p424-ADH-HA plasmids were modified with 59XhoI and 39SfiI sites downstream of the ADH promoter. To construct directional libraries, the first-strand cDNA syntheses were initiated with a NotI-oligo(dT) primer adaptor or a NotI- random hexamer primer adaptor for the p424-GAL1 and p424- GAL1-HA, and with a SfiI-oligo(dT) primer adaptor or a SfiI- random hexamer primer adaptor for the p424-ADH and p424- ADH-HA, according to the manufacturer’s instructions (Invitrogen). SalI adapters were ligated to the resultant double- strand cDNAs prior to digestion with NotI or SfiI. The cDNAs with SalI-NotI termini or with SalI-SfiI termini were ligated into the plasmid cloning vectors (XhoI-NotI-cut or XhoI-SfiI-cut, respectively). The number of initial transformants ranged from 1.36106 to 1.36107. A human B cell cDNA library was purchased from ATCC. The oligo(dT)-primed cDNAs were inserted into the XhoI cloning site, downstream of a GAL1 promoter. Procedures used for cDNA library screening are provided as Supplemental Information. Reporter gene assays. Plasmids were introduced into yeast by lithium acetate transformation. The yeast strain EGY48, which carries 6 lexA operators upstream of LEU2 gene (6op-LEU2), was transformed with pJK103 [10], which carries two lexA operators upstream of lacZ gene (p426-2op-lacZ), and subsequently with reporter plasmids (p413-TEF-Fas-d-S1-TA, p413-TEF-Fas-d-G1- TA, p413-TEF-Fas-d-S2-TA, p413-TEF-Fas-d-S3-TA, p413-TEF- Fas-d-S6-TA, p413-TEF-Fas-d-S8-TA, or p413-TEF-Fas-d-S9- TA). Caspase (all full-length zymogen proforms) expression Yeast-Based Protease Technlgys plasmids (p424-ADH-Caspase1-FLAG, p424-ADH-HA-Caspase2, p424-ADH-Caspase3, p424-TEF-Caspase4, p424-ADH-Caspase5, p424-TEF-HA-Caspase6, p424-ADH-Caspase7, and p424-ADH- HA-Caspase9-FLAG) or empty vector (p424-ADH), were intro- duced into these backgrounds. As for assays with Caspase-8 and -10, we expressed small amounts of pro-Caspase-8 and -10 with a large amount of FADD by transforming yeast with the dual gene plasmids p424-CYC1-Caspase8-HA/TEF-HA-FADD and p424-CYC1- Caspase10-FLAG/TEF-HA-FADD, because expression of large amounts of human Caspase-8 or Caspase-10 significantly inhibited the cell growth. For ATG4 assays, the yeast strain EGY48 was transformed with ATG4B or catalytic mutant ATG4B(C/A) plasmids (p424-TEF-ATG4B-FLAG or p424-TEF-ATG4B(C/A)- FLAG), reporter plasmid (p413-TEF-Fas-d-LC3B-TA), and pJK103 or pSH18-34 [10], which carries eight lexA operators upstream of a lacZ gene. The transformants (two independent colonies for each transformation) were streaked on growth plates (minimum synthetic dropout (SD) medium containing 2% glucose and 50 mg/ml leucine) or on selection plates (SD medium containing 1% galactose, 0.2% raffinose, BU salts, and 80 mg/ml X-gal). Yeast growth and blue color development were monitored for four to six days at 30uC. Reporter gene assays in liquid media using 384-well plates. For caspase assays, the assays were performed in a total volume of 40 ml in triplicate. First, 20 ml of liquid selection media containing X-gal and a series of concentrations of reagents such as z-VAD was dispensed into each well of 384-well plates. Next, confluent yeast cells expressing various Caspases and cleavable substrates were collected, washed with water, and suspended in selection media of the same volume as the culture media. The yeast suspension was diluted to 1:5-10 (v:v) with the selection media, and 20 ml aliquots were added to the 384-well plates. For ATG4B assays, yeast cells expressing ATG4B and LC3 fused- transcription factor were cultured overnight in standard growth medium, then the yeast cells were diluted in selection medium (SD medium containing 1% galactose, 0.2% raffinose, BU salts, and 100 mg/ml X-gal) at 104/ml and dispensed 100 ml into each well of 384-well plates. Absorbance at 620 nm was measured 2–3 days after culture at 30uC. HTS Assays. For the Fas/FADD/Caspase-8 and the NLRP1/ Asc/Caspase-1 assays, EGY48 yeast containing the desired plasmids were streaked onto SD plates (6.8 g Yeast Nitrogen Base w/o amino acids, 20 mg arginine, 50 mg threonine, 30 mg isoleucine, 60 mg phenylalanine, 20 mg valine) containing agar (1.7%), supplemented with 2% a-D-glucose and 50 mg/mL leucine. The plates were incubated at 30uC for 48 hrs and a colony was picked and transferred to a 14 ml polypropylene tube containing 2 ml of SD media broth supplemented with a-D-glucose and leucine as above and grown at 30uC for 16–24 hrs with shaking. Then, 1 ml of the overnight culture was transferred into 20 mls of growth media in a 500 ml flask and shaken at 30uC for 16–24 hrs. The cells were collected by centrifugation at 1000xg for 5 minutes at room temperature, and washed with 20 mls of sterile water, then resuspended in 20 mls of SD broth supplemented with 1% galactose and 0.2% raffinose. The HTS Assay was performed at a final compound concentration of 10 mM (1% DMSO), with cell densities of 26105 cells/ml (26105 cells) in a volume of 40 ml. The assay was assembled by the addition of 4 ml of <100 mM compounds (final concentration of 10 mM) in 10% DMSO (1% final DMSO) to clear polystyrene 384-well microplates using a Beckman-Coulter Biomek FX, then 18 ml of cell suspension (1.16107 cells/ml) (26105 cells/well) and 18 ml of X-gal suspended in Selection Media (for a final concentration of 400 mg/mL) were added to the wells using a Matrix WellMate bulk reagents dispenser. Remaining PLoS ONE | www.plosone.org 13 October 2009 | Volume 4 | Issue 10 | e7655