Cbf11 likely affects stress-gene expression indirectly.

(A) Western blot analysis of Atf1 and Sty1 phosphorylation in WT, sty1Δ, and cbf11Δ cells treated or not with 1 mM H₂O₂ for 15 min in YES medium. Atf1 phosphorylation manifests as retarded migration through the gel. (B) Recruitment of Atf1 to the indicated stress-gene promoters was analyzed by ChIP-qPCR in untreated cells grown in EMM medium. Mean and SD values of two independent replicates are shown. Two-sided Mann-Whitney U test was used to determine statistical significance. (C) Pap1-GFP localization was observed in formaldehyde-fixed cells grown to exponential phase in YES using epifluorescence microscopy. Cells were either untreated (control), treated with 0.2 mM H₂O₂ for 25 min as a stressed control or treated with 20 μM cerulenin for 10 min and 1 hour to inhibit FA synthesis. Scale bar 5 μm. (D) Recruitment of Cbf11 to the indicated stress-gene promoters was analyzed by ChIP-qPCR in cells treated or not with 0.5 mM H₂O₂ for 30 min in YES. The cut6 promoter is a positive control for Cbf11 binding [17];”control” is an intergenic locus with no Cbf11 binding (Chr I: 1,928,359–1,928,274). Mean and SD values of three independent replicates are shown. One-sided Mann-Whitney U test was used to determine statistical significance; since no stress promoter loci showed signal higher than the negative control locus, these loci were not tested statistically. (E) Survival and growth under oxidative stress of WT, cbf11Δ, cbf11-TAP, and cbf11DBM-TAP cultures spotted on YES plates containing 1.5 mM H₂O₂.