530 emission filter. Cy3 and Cy5 were visualized using 543 nm excitation/BP 560–615 nm emission and 633 nm excitation/ 650 nm LP filter respectively. DAPI was visualized using 405 nm excitation and 470–500 nm emission BP filters. Reporter assays Transcriptional activation of HSF1 was assayed using an hsp70.1-based luciferase reporter construct as described previously [35]. Results were normalized to b-galactosidase expressed from a co-transfected pCMV-LacZ plasmid [36]. Chromatin Immunoprecipitation ChIP assays for HSF1 association with chromatin were performed essentially as described previously [37]. ChIP was carried out with anti-HSF1 antibodies. Pre-immune IgG ChIP was used as a negative control and HSF1 binding data were normalized to this IgG control. The genomic DNA (of 1% of starting lysate/Input) was used as a positive control and pre- immune IgG mock ChIP as negative control. Amplified PCR products were first analyzed by size on agarose gel. In addition the PCR products were independently quantitated by using ABI 7300 real time PCR system and the -fold increase in ChIP-PCR products by 2-DDCT compared with the control (pre-immune IgG) was plotted for the respective region of hsp70.1. ChIP experi- ments and PCR amplifications were performed 3 times for each sample. In vitro kinase assay and mass spectrometric analysis of phospho-HSF1 Human HSF1 was cloned into the pGEX-2T vector and then grown up in BL21 (DE3) E. coli cells. The resulting GST-tagged HSF1 was isolated on a glutathione column and eluted according to the manufacturer’s specifications (Pierce). For in vitro kinase assay, GST-HSF1 was incubated with 3 units of recombinant PKAca, 1 mM ATP and 1X kinase assay buffer (Cell Signaling) at 37uC for 2 hr then 30 minutes at 43uC. The reaction was terminated by adding Laemmli sample buffer. For the mass spectrometric analysis, the samples were run on a 7% SDS-PAGE gel, stained with Coommassie Blue R-250 and a band greater than 85 kDa was cut out along with a ‘‘blank’’ band from another lane. After trypsin digestion, polypeptides were analyzed at the Taplin Mass Spectrometry Facility, Harvard Medical School, using an LTQ-Orbitrap (Thermo Electron). Cyclic-AMP assay Cyclic-AMP concentrations in cell lysates were assayed according to manufacturer’s instruction using an Assay Designs cyclic AMP Enzyme Immunoassay (EIA) kit. This is a competitive immunoassay for quantitative determination of cAMP. Levels of cAMP in HeLa cells were normalized to 100% and quantitation of cAMP was done with respect to 100% cAMP in control HeLa cells. For experiments to determine the role of cAMP in HSF1-PKA binding, some cells were pre-treated with the adenylate cyclase inhibitor t-busate at a concentration of 200 nM for 30 minutes. Results (1) HSF1 binds PKAca Association between HSF1 and PKAca was first indicated in the pulldown analysis mentioned above, using GST-HSF1, HeLa cell lysates, mass spectrometry and database analysis [34] (Fig. S1). We confirmed that these proteins interact in vivo using HSF1 and Protein Kinase A immunoprecipitation of PKAca from HeLa cell lysates by anti- PKAca antibodies followed by immunoblot analysis with anti- HSF1 antibodies in HeLa cells (Fig. 1 A). Intracellular HSF1 was co-immunoprecipitated by these antibodies in association with PKAca and this interaction was also observed after heat shock. We next examined whether HSF1 can bind to regulatory subunits from the PKA holoenzyme (PKA R1 or PKA RII) using co-immunoprecipiation analysis with anti-PKA-R1 and anti-PKA-RII antibodies (Fig. 1A). Trace levels of HSF1 binding to PKAR1 were observed after heat shock although we did not detect association with PKARII (Fig. 1A). However, the intracellular levels of PKA-R1 and PKA-RII were considerably lower than the concentration of PKAca by the criterion of intensity of the immunoblot bands and preferential association may reflect this higher concentration (Fig. 1A). Nonetheless, these data are consistent with the original proteomic screen in which we found strong evidence for HSF1 binding to PKAca but did not detect interaction of GST-HSF1 with the PKA regulatory domains. We next carried out the inverse analysis using immunoprecip- itation with anti-HSF1 antibodies followed by immunoblot with anti-PKAca antibodies. Because HSF1 can also be activated by a number of other agents including the Hsp90 inhibitor 17AAG, the proteasome inhibitor MG-132 and growth factors such as heregulin (HRG) and insulin-like growth factor (IGF-1), we also examined the effect of these molecules on HSF1-PKAca association. For these experiments we used MCF-7 breast carcinoma cells as these cells respond vigorously to the growth factors. We observed a significant basal level of HSF1-PKAca binding in these cells and association was strongly increased by HRG and MG-132 (Fig. 1B). Heat shock did not cause a major increase in HSF1-PKAca complex formation in the MCF-7 cells. However, heregulin and MG-132 led to major increases in PKA co-association (Fig. 1B). When we examined HSF1 in the lysates, we found that heat shock led to electrophoretic retardation of HSF1, consistent with stress-induced hyperphosphorylation shown previously. PKAca levels were largely unaltered. We also measured the levels of an HSF1 product (Hsp70). Hsp70 concentrations were increased by each treatment (Fig. 1B). We then carried out the reverse procedure in MCF-7 cells, immunoprecipitating with anti-PKAca antibodies and observed the co-precipitation of HSF1 with immunoprecipitated PKAca (Fig. 1C). HSF1 levels were consistently increased in cells treated with HRG and HSF1-PKAca association appeared to increase in each case (Fig. 1 B, C). This was not observed for the other treatments in which HSF1-PKAca association was maintained after the treatments but not consistently increased (Fig. 1A–C). PKAca levels were not consistently altered by any of the treatments and changes in levels of the kinase do not appear to influence responses to HSF1 activators (Fig. 1, A–C). As PKA can be induced through the activation of adenylate cyclase, we examined a scenario in which HSF1-binding to PKAca is regulated through the product of adenylate cyclase activity, cAMP (Fig. 1D). Indeed, using the adenylate cyclase inhibitor t- busate, we observed a reduction in HSF1-PKAca binding, a decrease that correlates with lower cAMP levels under these conditions, suggesting a role for cAMP in the interaction. However, we did not detect an increase in intracellular cAMP levels after 1 hour of heat shock at 43uC in these cells (Fig. 1D, right panel). This could be interpreted as meaning that basal levels of cAMP are required to maintain a pool of free intracellular PKAca capable of HSF1 binding (Fig. 1D). We next measured cAMP levels in cells treated with the other activators HRG, IGF1, 17AAG and MG-132. Once again we did not detect an increase in PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e13830