strain from which the deletion collection was derived (BY4743, MATa/a his3D1/his3D1 leu2D0/leu2D0 lys2D0/LYS2 MET15/ met15D0 ura3D0 /ura3D0 ORF::kanMX4) was used to determine the dose of compound that resulted in 15% growth inhibition. Cells were inoculated at an OD600 of 0.0625 in serial dilutions of drug and grown in a Tecan GENios microplate reader (Tecan Systems Inc., San Jose, USA) at 30uC with orbital shaking. Optical density measurements (OD600) were taken every 15 minutes until the cultures were saturated, and doubling time (D) was calculat- ed as described [47]. For genome-wide fitness profiles, ,4700 homozygous deletion strains and ,1100 essential heterozygous deletion strains were assayed as described [46] combining 5- generation homozygous pools and 350 ml from the 20-generation heterozygous essential pool prior to genomic DNA extraction. 200 ng of genomic DNA was added to 2 separate PCR reactions, one each with primers designed to amplify all UPTAGs and DOWNTAGS. One primer in each reaction was biotinylated such that it could be detected following hybridization using streptavi- din-phycoerythrin. Intensity values for the probes on the chip were extracted using the GeneChip Operating Software (Affymetrix). Quantile normalization, outlier omission, fitness defect ratios performed as previously described [47]. The larger the ratio, the more depleted (sensitive) is the strain as compared to control condition without the drug. Supporting Information Figure S1 Plate halo assay: Compound 4a, 4b, and SB- 203580. SB203580 does not enter yeast cells. A lawn of yeast cells that lack the major multidrug export protein Pdr5 (pdr5D) was spread on solid medium in the absence (control) or presence of osmotic stress (1.5 M sorbitol), and filter discs containing various concentrations of 4a, 4b, and SB203580 were placed on top of the lawn. Inhibition of Hog1 activity can be visualized by the formation of a halo of non-proliferating cells around the filter discs in the presence of osmotic stress (1.5 M sorbitol). No such halo is visible on control plates. Plates were incubated for 48 hours at 30uC. (TIF) Figure S2 Plate halo assay: Commercially available p38 inhibitors. Commercially available p38-inhibitors do not enter yeast cells. A lawn of wild type yeast cells was spread on solid medium in the absence (control) or presence of osmotic stress (1.5 M sorbitol), and filter discs containing 2 mM of the indicated inhibitors were placed on top of the lawn. Inhibition of Hog1 activity can be visualized by the formation of a halo of non- proliferating cells around the filter discs in the presence of osmotic stress (1.5 M sorbitol). No such halo is visible on control plates. Plates were incubated for 48 hours at 30uC. (TIF) Figure S3 Phosphorylation of a kinase-dead Hog1 allele (Hog1K52R). Sustained phosphorylation of a kinase-dead Hog1 allele (Hog1K52R) in response to osmotic stress. hog1D cells were transformed with a plasmid containing the kinase-dead Hog1K52R allele. Phosphorylation was monitored in cells exposed to osmotic stress (0.4 M NaCl) by western blot analysis using an antibody specific to dually phosphorylated p38 MAPK, and an anti-Hog1 antibody as a control. (TIF) References A Hog1 Inhibitor for Analyzing Kinase Signaling Figure S4 Inhibition of Hog1-dependent gene expres- sion (STL1-lacZ). Inhibition of Hog1-dependent gene expres- sion. Exponentially growing cells harboring the STL1-lacZ reporter were exposed to osmotic stress (0.8 M sorbitol) and assayed for b- galactosidase activity as described in the Experimental section. Induced expression of the STL1 gene by osmotic stress requires Hog1. 4b was added to cells at the indicated concentrations 10 minutes before osmotic stress was applied. The results are the average of three independent experiments and the error bars represent standard deviation (s.d.). (TIF) Figure S5 Hog1 kinase activity required to relieve As(III)-induced G1 checkpoint arrest. Hog1 kinase activity is required to relieve As(III)-induced G1 checkpoint arrest. (A) HOG1 deletion or addition of 4a result in persistent G1 arrest in the presence of As(III). (B) As(III)-induced G1 checkpoint delay can be prolonged by addition of 4a until just before onset of the S phase. (C) Removal of 4a quickly relieves G1 arrest. Wild-type (W303-1A) and the isogenic HOG1 deletion mutant (hog1D) were synchronized in G1 with 5 mM a-factor and released in fresh medium in the presence or absence of 0.5 mM sodium arsenite [As(III)]. 4a (1 mM) was added as indicated. After washing out the inhibitor (in C), the cells were resuspended in fresh medium containing 0.5 mM As(III). The percentage of cells that remained in G1 was determined by the a-factor-nocodazole trap assay. (TIF) Figure S6 4a improves growth of PBS2 and SSK1 overexpressing cells. 4a improves growth of PBS2 and SSK1 overexpressing cells. Wild-type and hog1D cells (BY4743 strain) were transformed with an empty plasmid or plasmids overex- pressing PBS2 or SSK1. Cells were grown in a micro-cultivation system in the absence or presence of inhibitor as indicated. (TIF) Figure S7 4b improves growth of PBS2 and SSK1 overexpressing cells. 4b improves growth of PBS2 and SSK1 overexpressing cells. Wild-type and hog1D cells (BY4743 strain) were transformed with an empty plasmid or plasmids overex- pressing PBS2 or SSK1. Cells were grown in a micro-cultivation system in the absence or presence of inhibitor as indicated. (TIF) Table S1 Selectivity. Chemical genetic profiling of the yeast deletion mutant collection in the presence of 500 mM 4a. (XLS) Acknowledgments We thank Jeremy Thorner, Francesc Posas, Marcus Krantz, and Vlado Reiser for providing strains and plasmids. Author Contributions Conceived and designed the experiments: M. Gr/otli SH MT RW PD CN GG. Performed the experiments: PD JVV JK M. Gebbia TA IM. Analyzed the data: PD JVV JK M. Gebbia TA IM M. Gr/otli SH MT RW CN GG. Contributed reagents/materials/analysis tools: PD JVV JK M. Gr/otli TA IM. Wrote the paper: M. Gr/otli MT SH PD. 1. Goldstein DM, Gray NS, Zarrinkar PP (2008) High-throughput kinase profiling as a platform for drug discovery. Nature Reviews Drug Discovery 7: 391–397. 2. Specht KM, Shokat KM (2002) The emerging power of chemical genetics. Current Opinion in Cell Biology 14: 155–159. PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e20012