3. Statistical analyses were performed with graphpad Prism software version 5.00 (GraphPad Software, San Diego, CA). Unless otherwise Cyclopamine concentration indicated, the threshold level chosen for comparison of means was P < 0.01 by Student's t-test (one-tailed, nonpaired, equal variance), corrected for multiple comparisons (Šidák, 1967). To test the sensitivity

of the double mutant to different stressors, WT, Δchap1, Δskn7, and Δchap1-Δskn7 (ΔΔ) were grown on solid CMX containing either 0.75 M sorbitol – hyperosmotic stress, 0.4 M KCl – hyperosmotic and salt (ionic) stress, 20 mM H2O2 – oxidative stress, 30 μM menadione – superoxide stress or 25 mM CWS – interference with cell wall integrity (Ram & Klis, 2006) (Fig. 1). Growth of Δchap1, Δskn7, and ΔΔ in the presence of 20 mM H2O2 was completely inhibited compared with WT which showed about 40% growth relative to control (solid CMX without additives). On 0.4 M KCl, growth of the ΔΔ mutant was also inhibited compared with WT, but not completely, and it showed similar growth rate to the Δskn7 mutant. On 0.75 M sorbitol, the double mutant showed almost

complete inhibition, but again similar to the Δskn7 mutant; WT and Δchap1 were also inhibited, Δchap1 more than WT, but both less than Δskn7 and the double mutant. CWS affected the growth of Δchap1 (55%) and the double mutant (47%), whereas growth of the WT and Δskn7 was less inhibited (about 65%). On menadione, the double mutant was inhibited more than the WT and Δskn7 but as much as Δchap1 (Fig. 1a). 17-AAG The double mutant (ΔΔ) is sensitive to oxidative and osmotic stresses, as well as to stressors that compromise cell wall integrity, but to the same extent as each single mutant, and there is no evidence for an additive effect on inhibition of growth. The only additive effect on growth rate was found with

the cell wall stressor CWS, where the double mutant was more sensitive than either single mutant (significant at P < 0.01). WT and the mutants were also grown on liquid CMX with lower concentrations of hydrogen peroxide (0.625–10 mM) Niclosamide to test whether the double mutant is more sensitive than each single mutant (Fig. 1b). WT grew normally on all concentrations; apparently at these oxidant levels, WT can overcome the stress by expression of antioxidant genes, as shown previously (Lev et al., 2005). All three mutants show lower growth percentages than WT, but similar to each other. We tested the expression of antioxidant genes shown previously (Lev et al., 2005) to be under regulation by the transcription factor ChAP1: glutathione reductase, GLR1; thioredoxin, TRX2; thioredoxin reductase, TRR1; γ-glutamylcysteine synthetase, GSH1. In addition, we followed the expression of a superoxide dismutase gene, SOD1, and three catalase-encoding genes CAT1,2,3 (Robbertse et al., 2003).