Overexpression or depletion of <i>HSF1</i> induces filamentation.

<p>a) <i>HSF1</i> levels can be overexpressed to different levels by placing one or both alleles of <i>HSF1</i> under the control of the tetracycline-repressible promoter, <i>tetO</i>. Strains were grown in the absence of doxycycline (DOX) to mid-log phase. <i>HSF1</i> transcript levels were measured by quantitative RT-PCR and normalized to <i>ACT1</i> and <i>GPD1</i>. Data are means +/- standard error of the means for triplicate samples. *** indicates P value <0.005, unpaired t test. b) Overexpression of <i>HSF1</i> can bypass the temperature requirement for morphogenesis. Strains were grown in the absence of DOX for 4 hours at the indicated temperature. c) The <i>tetO-HSF1-TAP/tetO-HSF1</i> strain can be used to monitor the effect of both <i>HSF1</i> overexpression and depletion. <i>HSF1</i> is overexpressed in the <i>tetO-HSF1-TAP/tetO-HSF1</i> strain when grown in the absence of DOX (No DOX). <i>HSF1</i> expression is lowered when the strain is grown in the presence of 0.1 μg/mL DOX (Low DOX), and is depleted when grown with 20 μg/mL DOX (High DOX). <i>HSF1</i> transcript levels were measured by quantitative RT-PCR and were normalized to <i>ACT1</i> and <i>GPD1</i>. Data are means +/- standard error of the means for triplicate samples and are graphed on a log₁₀ scale. *** indicates P value <0.005, * indicates P value <0.05, unpaired t test. d) Hsf1 protein levels are overexpressed when the <i>tetO-HSF1-TAP/tetO-HSF1</i> strain is grown in the absence of DOX, reduced when grown in 0.1 μg/mL DOX (Low DOX, labelled L), and depleted when grown in 20 μg/mL DOX (High DOX, labelled H). WT indicates the wild type, untagged control. Tubulin levels serve as loading control. e) <i>HSF1</i> overexpression or depletion induces filamentation in the absence of an inducing cue. Strains were grown in the presence of no DOX, 0.1 μg/mL DOX (Low DOX), or 20 μg/mL DOX (High DOX) at 30°C.