Figure 4

(A) Thermotolerance assay. The transformed (URA+) cells, containing empty vector pRep42 (WT) or human GRα expressing vector (GRα) were subject to pre-treatment for 1 h at 37 °C, or not before the heat shock at 50 °C for 20 minutes. Yeast cultures were then serially diluted (serial multiple of ten-fold) before spotting on minimal selective media with or without DEX, and incubated for 5 days at 30 °C. Results are representative of three independent experiments. (B) Quantitative RT-PCR for Hsp104 gene. The level of expression of Hsp104 mRNA from control yeast (WT) and yeast expressing human GRα (GRα) was measured by qRT-PCR. The housekeeping gene Act was used as an internal control for gene expression. (C) Thermotolerance assay for ∆Hsp104 strain. ∆Hsp104 strain was transformed empty pRep42 plasmid or GRα expressing vector. Transformants were again subject to the thermotolerance assay. (D) Insoluble and soluble cytosolic fractions were separated by high-speed centrifugation before the fractions were resolved SDS-Page gel. Western blot analysis shows total, soluble and insoluble fractions probed with anti-GR antibody. (E) Western blot analysis of Sty1. Control yeast (WT) and yeast expressing the GRα were grown to an OD595~0.5. Extracts of WT and GRα cells were separated by SDS-PAGE, and probed for total Sty1 using a polyclonal antibody raised to the S. cerevisiae Sty1 homolog Hog1. For phosphorylated Sty1 we used the human anti- phospho p38 antibody. Equal loading was measured using an anti Actin antibody. Blots images have been cropped in order to improve the clarity of an effect of phosphorylation. Full images of gels see in Supplementary Fig. 1. (F) Sty1 protection from thermal stress. S. pombe strain JP198 (∆sty1) was transformed either with a control plasmid, pRep 41, or a human GRα expressing plasmid. Yeast were grown to OD595 ∼ 0.5 and then serially diluted 10 fold, and spotted on minimal media before 5 days at 30 °C or 39 °C. Results are representative of three independent experiments. (G) Summary schema of the two major regulatory pathways leading to Sty1 activation: a Ras1 pathway and Wak1/Win1 pathway. Ras1 has been shown to regulate two downstream pathways; one of which is the Byr2/Byr1/Spk1 kinase cascade and the other is the Byr2/Zfs1/Mra1 and Sty1 kinase cascade. Red arrows indicate connections identified within the GR interactome.