3H, J = 6.8 Hz). 13C NMR: d 162.6 (d, C-F J = 248 Hz), 158.4, 149.5, 144.0, 144.0, 137.4, 135.1, 132.0, 128.9, 128.8, 128.7 (d, C-F J = 7.7 Hz), 128.3, 127.6 (d, C-F J = 3.1 Hz), 127.5, 126.5, 125.8, 115.6 (d, C-F J = 21.5 Hz), 113.5, 107.2, 52.2, 52.1, 24.4. HRMS [M+1]+ calculated for C28H25FN5+: 450.2089; found 450.2087. Yeast strains and plasmids The following Saccharomyces cerevisiae strains were used: BY4741 wild-type (MATa his3D1 leu2D0 met15D0 ura3D0), BY4741 pdr5D::KanMX, BY4741 hog1D::KanMX, BY4743 (MATa/MATa his3D0/his3D0 leu2D/leu2D0 met15D0/MET15 LYS2/lys2D0 ura3D0/ura3D0), W303-1A (MATa, leu23/112 ura31 trp11 his311/ 15 ade21 can1100 GAL SUC2), YSH818 (W303-1A hog1D::LEU2), W303-1A-STL1-lacZ (MATa, leu23/112 ura31 trp11 his311/15 ade21 can1100 GAL SUC2 STL1-lacZ-URA3), and SO329 (MATa can1 his4 leu2 trp1 ura3-52 FUS1-lacZ-LEU2). The plasmid containing GFP-tagged Hog1 was constructed by digesting the plasmid pRV-65wt [41] with BamHI and HindIII and cloning the resulting 2.5 kb fragment containing HOG1-GFP into YCplac33 (CEN, URA3). The gToW plasmids pSBI40, gToW-PBS2 and gToW-SSK1 have been described previously [32]. Plate halo assay Wt and pdr5D cells were grown in YPD (1% yeast extract, 2% peptone, 2% glucose) medium until mid-log phase and mixed with low melting agarose and YPDA (YPD supplemented with 0.3% adenine) with or without 1.5 M sorbitol. The cell mixture was poured onto YPDA plates with or without 1.5 M sorbitol and allowed to solidify. Filter discs soaked with 5 ml inhibitor (or DMSO for the controls) in various concentrations were placed on top of the lawn of cells. The plates were incubated at 30uC and growth was scored after 1–2 days. Liquid medium micro-cultivation Yeast strains (BY4743 background) were grown for 72 hours in the presence or absence of the indicated concentrations of inhibitor using a high-resolution micro-cultivation approach as previously described [32,42]. Wt and hog1D cells were transformed with the empty gToW plasmid pSBI40 or the same plasmid overexpressing PBS2 or SSK1 [32]. Cell extracts and immunoblotting Wild-type cells were grown in YPD to an OD600 of approx- imately 1. The cultures were then incubated with the inhibitor, or the corresponding volume of DMSO as a control, for 15 minutes before applying the osmotic stress. Sorbitol, dissolved in YPD, was added to the cultures to a final concentration of 0.8 M. The corresponding volume of YPD was added to the control cultures. Samples (1 ml) were taken at 0, 15, 30, 60, and 120 minutes, spun down, resuspended in 50 ml protein extraction buffer (0.1 M Tris Cl, pH 6.8; 4% SDS; 20% glycerol; 0.2 M DTT; 10 mM NaF; 0.1 mM Na3VO4; protease inhibitor cocktail; 0.2 M b-mercapto- ethanol), and boiled for 10 minutes. Proteins were separated on a 7.5% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was saturated in Odyssey blocking buffer (Li-Cor Biosciences, Lincoln, NE) for 45 minutes at room temperature and then incubated overnight at 4uC with a rabbit monoclonal anti-phospho-p38 antibody (Cell Signaling Technol- ogy, Danvers, MA). After 365 minute washes in TBS (138 mM NaCl, 2.7 mM KCl, 5 mM Tris base) with 0.1% Tween, the membrane was incubated with a goat polyclonal anti-Hog1 (yC- 20) antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) for one hour at room temperature. The membrane-bound antibodies A Hog1 Inhibitor for Analyzing Kinase Signaling were detected with secondary IRDye 800CW donkey anti-rabbit and IRDye 680 donkey anti-goat antibodies (Li-Cor Biosciences, Lincoln, NE) and visualized with an Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE). Measurement of reporter gene expression Exponentially growing cells harboring either the STL1-lacZ or FUS1-lacZ reporter constructs were pretreated with the inhibitor or DMSO for 10 minutes followed by the addition of 0.8 M sorbitol or 10 mM a-factor. Cells were harvested, and b-galac- tosidase activities of the protein extracts were assayed according to the literature [43]. In vitro Hog1 kinase assays Kinase assays were performed in 384-well plate format using purified GST-tagged Hog1 and the biotinylated peptide (biotin)- DVPG-T-PSDKVITF as a substrate, where the peptide se- quence corresponds to the sequence in Sic1 that is targeted and phosphorylated by Hog1. Hog1 was activated in vitro using a constitutively active upstream kinase (GST-Pbs2EE), as described previously [14,44]. To the reaction wells, 2 ml of the inhibitor in 10% DMSO were added at the desired concentrations; the final concentration of DMSO in all reactions was 1%. A kinase reaction master mix was prepared and added in 18 ml aliquots to the wells; thereafter the plate was incubated in a water bath for 4 hours at 37uC with agitation. The final concentrations in the reactions were: 0.02 mg/ml active Hog1, 100 mM substrate peptide, 0.2 mM ATP, and 0.1 mCi/nmol [32cP]-ATP in a kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT). After incubation, 2 ml of each reaction was transferred to a SAM2 membrane (Promega, V7861), which was washed and dried as previously described [45]. The membrane was then exposed to a Phosphor- Imager screen and subsequently imaged in a Molecular ImagerH FX (BioRad). To quantify the kinase activity in the assays, the density of the signal from each spot on the membrane was determined (QuantityOne-4.6.8; Bio-Rad Laboratories). For the inhibition curves (Figure 4B), the initial sample of each series (0.1 nM inhibitor) was set to 100% activity, whereas in the comparative study (Figure 4A), a reference reaction with 1% DMSO was used as a sample. For the calculation of the IC50 values, a four-parameter model in BioDataFit 1.02 (www. changbioscience.com) was used. Cell cycle experiments To analyze the G1/S checkpoint during As(III) stress, yeast cells synchronized in G1 with 5 mM a-factor were released into fresh YPDA medium containing 0.5 mM As(III) in the presence or absence of 1 mM of inhibitor. In control experiments, G1-arrested cells were released into a medium lacking As(III) in the presence or absence of 1 mM of inhibitor. Samples were collected at 20-minute intervals for the a-factor-nocodazole trap assay to determine the percentage of cells that remained in G1, and at 30-minute intervals for Western blotting to monitor dually phosphorylated and total Hog1. The a-factor-nocodazole trap assay, protein extracts, and Western analysis were performed as described [20]. All cell cycle experiments were repeated at least twice, and representative results are presented. Chemical Genetic Profiling Screens were performed essentially as described by Ericson et al, [46]. Compound doses for the genome-wide screens were determined by performing a dose response for each compound and 2% DMSO to serve as the vehicle control. The wild-type PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e20012