Impact of stepwise heat shocks.

<p>The model was used to simulate stepwise thermal transitions from 30°C (the starting condition) to 37°C, and then from 37°C to 42°C. Outcomes were then tested experimentally by determining Hsf1 phosphorylation levels. (A) Representations of the thermal transitions. (B) Model simulation of Hsf1 phosphorylation during stepwise 30°C–37°C and 37°C–42°C heat shocks. (C) These predictions were tested experimentally by moving exponentially growing cells from 30°C to 37°C for 30 minutes, and then transferring them from 37°C to 42°C for 30 minutes. Proteins were extracted at various time intervals and Hsf1 phosphorylation measured by western blotting. Hsf1 was shown to be transiently induced during the 37°C–42°C transitions. Lambda phosphatase (λ Ppase) controls were included to confirm that Hsf1 phosphorylation had returned to basal levels 30 minutes after a 30°C–37°C heat shock (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032467#pone-0032467-g002" target="_blank">Figure 2</a>), but that Hsf1 is rapidly phosphorylated following the next 37°C–42°C heat shock. The data reflect the outcomes for at least two independent replicate experiments.