HSP70 Transcription and Protein Aggregation Figure 4. PKA and CDK9-dependent resolution of poly Q-GFP protein aggregates during heat shock. (A and B) Poly-103Q-GFP expressing PC12 cells were grown at 37uC and subjected to sham heating (A) or 42uC for 1 hr (B) before staining for HSF1 and DAPI and analysis by confocal microscopy. (C and D) Cells were transfected with PKAca siRNA for 72 hours then sham heated (C) or incubated at 42uC for 1 hr (D) and analyzed by confocal microscopy for Poly-Q-GFP and PKAca. (red) (E and F). Cells were transfected with CDK9 siRNA for 72 hours, treated +/2 42uC then stained for CDK9 using anti CDK9 antibodies. Poly-Q-GFP and CDK9 (red) are analyzed by confocal microscopy. Experiments were carried out 3 times, reproducibly. (G) Cells with aggregate bodies were counted in cells transfected with siRNA for HSF1 (image is not shown) and PKA (C and D), each compared to scrambled control RNA and plotted. These data are the average of three independent experiments 6 SEM. (H) HeLa cells were transfected with either Htt 94Q-CFP or Htt 25Q-CFP, for 24 hr and subjected to sham heating or 42uC for 1 hour. Cells were collected and then lysates were diluted to equalize total protein concentration and subjected to centrifugation at 400 g for 10 min. (P, pellet and S, supernatant). PC12 cells expressing 103Q-GFP were transfected with or without HSF1 siRNA, HSF1 scr, PKA siRNA, PKAscr for 72 hours and then subjected to sham heating or 42uC for 1 hr. Cell lysates were collected as for HeLa cells and centrifuged at 400 g for 10 mins. The pellet and supernatant fractions were run on 4– 15% gradient SDS-PAGE and immunoblotted with anti GFP Ab. (I) Schematic representation of PKAca-dependent transcription of hsp genes and resolution of protein aggregates. doi:10.1371/journal.pone.0028950.g004 encoding hsp70.1 and NsB-like foci containing large non-coding RNAs after stress (Fig. 1, 4I). NsB, contain long tandem arrays of Sat III repeats that bind HSF1 and give rise to non-protein coding transcripts [15,14]. HSF1- PKAca interaction may thus have a versatile role in governing expression both of the cohort of HSF1- induced mRNAs and non-coding RNAs after stress. This pathway appears to be involved in folding of poly-Q containing, insoluble protein inclusion bodies during activation of HSF1 and depletion of either PKA or CDK9 inhibits this effect, presumably through reduction in HSP-mediated folding (Fig. 4). Accumulation of protein inclusions is associated with neurode- generative disorders such as Huntington’s disease that manifests during aging and may result in triggering of death pathways and decline in neuronal networks [3]. HSF1 is a potent factor in the refolding of poly-Q containing protein aggregates [27], (Fig. 4). Our delineation of a pathway for HSF1 activation and resolution of protein aggregates, involving PKAca may thus suggest novel avenues to investigate age-dependent decline in HSF1 activity. In PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28950