HSF1 and Protein Kinase A Figure 3. Intracellular location of PKAca under HSF1 in different activation conditions. A–D) MCF-7 cells were plated and treated without and with heat shock at 43uC for 1 hour (A, B). Cells were also treated with 30 mM HRG (C) and 100 ng/ml IGF-1 (D) for 24 hours. Cells were then fixed and probed for PKAca and HSF1 localization with anti- PKAca and anti-HSF1 antibodies. Cells were later stained with fluorophore conjugated secondary antibodies goat anti-rabbit Alexa 488 (for PKAca, green) and goat anti-rat Cy3 (for HSF1, red) antibodies. Nuclei were visualized with DAPI (nucleus, blue). In experiments A–D, co-localization is indicated with arrows in merge images E) Numbers of cells with partial or complete co- localization of HSF1 and PKAca were counted for three independent experiments. The values for treated cells (heat, HRG, IGF1) are significantly different from non-treated control cells at p,0.001 (Student’s t-test) under each condition. F,G) HeLa cells were infected with PKA shRNA (kd #1) and then incubated (G) at 43uC for 1 hour or sham heated but maintained at 37uC (F). Cells were stained with anti-PKAca and anti-HSF1 antibodies. For indirect immunofluorescence, cells were stained with secondary antibodies; goat anti-Alexa 488 (PKAca, green) and goat anti-rat Cy3 (for HSF1, red). Nuclear staining (DAPI) is also shown for these cells. Scale bar 5 mm. Experiments (A–D, F, G) were repeated at least 3 times with consistent findings. doi:10.1371/journal.pone.0013830.g003 the phosphoserine. As the presence of wild-type HSF1 in such cells could complicate the interpretation, we carried these experiments in cells in which HSF1 was depleted by specific shRNA. We were unsuccessful in preparing stable HSF1 knockdown cell lines in either HeLa or MCF-7 as significant reductions in HSF1 were lethal to these cells (data not shown). We therefore used a prostate cancer cell line, DU-145 in which we were able to induce profound reduction in HSF1 levels by knockdown with shRNA and yet maintain viability. Inactivation of serine 320 by alanine substitution led to an HSF1 species refractory to heat-induced nuclear localization and which was detected in cytoplasmic foci (Fig. 4A). In cells expressing HSF1-GFP (S320D), this mutant form of HSF1 was localized to the nucleus after stress, indicating a role for negative charge at serine 320 in heat-induced nuclear PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e13830