Fig 2: Effects of HSF1 truncation and internal deletion mutants on hsp70 promoter-reporter activity and HSF1 dimerization. (A) Expression of TAD-deleted HSF1 constructs inhibits endogenous HSF1 driven hsp70 promoter-reporter activity, while removal of the HR-A/B domain abrogates dominant negative activity. HEK293 cells were transfected with hsp70 promoter-reporter construct and HSF1 C-terminal truncation constructs, treated with DMSO or 17AAG for 1 hour, heat shocked at 42 °C for 30 minutes and allowed to recover for 4 hours before harvesting. (B) Disruption of HA-tagged HSP90a E47A interaction with HSF1 C-terminal RD truncations by 17AAG. HEK293 cells were transfected with HA-HSP90a E47A and FLAG-HSF1 RD truncation mutants, then analyzed by anti-FLAG (HSF1) IP followed by HA (HSP90a E47A) WB. Pull-down experiments were repeated at least twice. (C) Deletion of the DBD and retention of the HR-A/B domain inhibits hsp70-reporter activity driven by endogenous HSF1. HEK293 cells were transfected, treated and processed as described in panel A. (D) Deletion of the HR-A/B domain disrupts HSF1 dimerization. HEK293 cells were transfected with WT HA-HSF1 and FLAG-HSF1 deletion constructs, then analyzed by anti-FLAG IP and HA WB. Pull-down experiments were repeated at least twice. (E) Summary of the effects of HSF1 domain deletions on hsp70-reporter activity. Full blots used to create cropped figure panels are shown in Supplementary Figure 6.

Fig 3: Mapping interaction sites of “closed” HSP90 on HSF1. (A) Schema of FLAG-HSF1 C-terminal truncation constructs and their expression in HEK293 cells. The associated blot shows expression of each construct in HEK293 cells. Equal amounts of DNA were used for transfection. For construct C477, the upper band represents the intact truncation (designated by an asterisk), while the lower band is likely a proteolytic fragment. (B) Schema of FLAG-HSF1 N-terminal truncation and internal deletion constructs and their expression in HEK293 cells. The associated blot shows expression of each construct in HEK293 cells. Equal amounts of DNA were used for each transfection. Note the poor expression of N195. For N195-C383, the lower band represents the intact construct (designated by an asterisk); the upper band may reflect a post translationally modified peptide. (C) Interaction of selected FLAG-HSF1 C-terminal truncation constructs with HA-tagged HSP90a E47A. HEK293 cells were transfected with either FLAG-HSF1 constructs or FLAG-pcDNA control and HA-HSP90a E47A, and then analyzed by anti-FLAG (HSF1) IP followed by HA (HSP90a E47A) WB. Pull downs were performed in triplicate and western blots analyzed by densitometry. All HA IP bands were normalized to their respective FLAG IP signals (see Supplemental Figure 6, compilation of full blots) and then normalized to the WT signal. A one-way ANOVA utilizing multiple comparisons was performed to determine significant differences in HSP90 interaction between truncated HSF1 constructs and F-HSF1 WT. A representative blot is shown above the bar graph. Significance is indicated by asterisks (**Indicates p < 0.01). The p-value for the difference between C339 and WT interaction was 0.0534. (D) Interaction of FLAG-HSF1 N-terminal truncation and internal deletion constructs with HA- tagged HSP90a E47A: HEK293 cells were transfected and analyzed as in panel C. Significance is indicated by asterisks (**Indicates p < 0.01 and ****Indicates p < 0.0001). Full blots used to create cropped figure panels and for densitometry analysis for the bar graphs are shown in Supplementary Figure 6.

Fig 4: HSF1 is bound by HSP90 in the “closed” conformation. (A) HSP90 ATP-driven conformational cycle. HSP90a E47A/HSP90ß E42A mutants bind ATP but cannot hydrolyze it and remain trapped in the “closed” N-domain dimerized conformation. Conversely, HSP90a D93A/HSP90ß D88A mutants do not bind ATP and remain trapped in an “open” N-domain undimerized conformation. (B) HSF1 interaction with HSP90 “closed” and “open” mutants. HEK293 cells were co-transfected with HA-HSF1 and with each FLAG-HSP90 construct, harvested and analyzed for HSF1/HSP90 association by anti-FLAG IP - HSF1 WB. Pull-down experiments were repeated at least twice. (C) Reproducible and reciprocal HSF1 interaction with “closed” HSP90a E47A and HSP90ß E42A mutants in the presence and absence of MoO4. Interactions were assessed by anti-FLAG (HSP90) IP - HSF1 WB (top panel) or anti-FLAG (HSF1) IP–HA (HSP90) WB (bottom panel). Pull-down experiments were repeated at least twice. (D) Effect of HSP90 inhibitors on HSF1-HSP90a E47A interaction. HEK293 cells were transfected with HA-HSF1 along with FLAG-HSP90a E47A, treated with N-terminal or C-terminal HSP90 inhibitors for 4 hours and analyzed by anti-FLAG (HSP90) IP- HSF1 WB. Pull down experiments were repeated at least twice. Full blots used to create cropped figure panels are shown in Supplementary Figure 6.

Fig 5: Impact of RD phosphorylation on HSP90 inhibitor effect on HSF1 activity and mobility shift in SDS-PAGE, and on HSF1 interaction with HSP90 (A) Schema of WT and dPRD FLAG-HSF1 constructs depicting RD phosphorylation sites mutated to alanine in dPRD-HSF1. (B) The N-terminal HSP90 inhibitor 17AAG enhances transcriptional activity of dPRD HSF1. HEK293 cells were transfected with hsp70-reporter construct and HSF1 C-terminal truncation constructs, treated with DMSO or 17AAG for 1 hour, heat shocked at 42 °C for 30 minutes and allowed to recover for 4 hours before harvesting. This experiment was repeated three times and values represent mean +/- S.D. (C) N-terminal HSP90 inhibitors induce a mobility shift in dPRD HSF1 similarly to that seen with WT-HSF1. HEK293 cells were transfected with WT or dPRD HSF1, treated with the three N-terminal HSP90 inhibitors as shown, and harvested and HSF1 was analyzed by WB. Full blots used to create cropped figure panels are shown in Supplementary Figure 6.