Interaction of HSF1 and HIF1α with HSP90 isoforms.

<p><b>(A)</b> HSF1 interaction with HSP90 WT and mutants: HEK293 cells were transfected with HA-HSF1 and each FLAG-HSP90 construct, harvested, immunoprecipitated with anti-FLAG beads and western blotted for HSF1 interaction. Input lysates were normalized and run as controls. <b>(B)</b> HIF1α interaction with HSP90 WT and mutants: HEK293 cells were transfected with HA-HIF1α and each FLAG-HSP90 construct, harvested, immunoprecipitated with anti-FLAG beads and western blotted for HIF1α interaction. Input lysates were normalized and run as controls. <b>(D)</b> Measurement of the relative interaction strength of HSF1 and HIF1α with each HSP90 isoform by LUMIER: HEK293 cells transfected with HSF1 or HIF1α and each HSP90 isoform were harvested, applied to a 96-well anti-FLAG plate and assayed for luciferase activity. The difference in relative interaction strength of HSP90α and HSP90β for HIF1α (>3-fold) was statistically significant (p<0.05); the difference in relative interaction strength for HSF1, while much less, was also statistically significant (p<0.05) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141786#sec008" target="_blank">Methods</a>). <b>(E)</b> Effect of each HSP90 isoform on heat shock induced gene expression: HEK293 transfected with each HSP90 isoform were heat shocked at 42°C for 30 minutes, allowed to recover for 2 hours, then harvested and assayed by qPCR for <i>HSPA1A</i>, <i>DNAJ1</i> and <i>STIP1</i> expression. <b>(F)</b> Effect of each HSP90 isoform on hypoxia-induced gene expression: HEK293 cells transfected with each HSP90 isoform were treated with 100 μM CoCl₂ for 2 hours, harvested and assayed by qPCR for <i>SLC2A1</i> and <i>VEGFA</i> expression.