STAT2, Caspase3 and HDAC6 as Hsp27 Client Proteins Figure 1. Transient down regulation of Hsp27 protein by shRNA sensitizes HeLa cells to cell death and promotes apoptosis. A, pCI- Neo, pSuperNeo-ScRNA27 (Sc27), pSuperNeo-MsRNA27 (Ms27) and pSuper-ShRNA27 (Sh27) vectors were transiently expressed in HeLa cells. Samples were collected 48 h after transfection and Hsp27 level was analyzed by western blot. Level of actin was used as control. B, Sensitivity to cell death was determined by crystal violet analysis, as described in Materials and methods. Staurosporine, a pro-apoptotic kinase inhibitor, was used to induce apoptosis (p,0.01). C, HeLa cells, treated for 4 h with different concentrations of staurosporine, were analyzed by western blot using an anti- caspase3 antibody. doi:10.1371/journal.pone.0029719.g001 depleted HeLa cells showed strong nucleus abnormalities. Nuclei were no more spherical and well shaped but morphologically affected. All cells of the highly depleted clone HSh27-2.2 presented nucleus abnormalities and giant cells bearing up to 20 nuclei were frequently observed (Fig. 3.B). This polyploidy confirmed the observation described in Fig. 2.D highlighting the drastic increase in G2 cells. Interestingly, an increase in plurinucleated cells was also detected by flow cytometry analysis in MCF-7 clones, but this increase was not associated with any alterations of nucleus phenotype (data not shown); this is probably due to the high level of Hsp27 which remains in these cells. Hsp27 is well referenced to support cytoskeletal integrity, particularly by protecting F-actin disruption in stress condition [29,30]. Consequently, we next performed immunofluorescence analysis of actin cytoskeleton of the different HeLa cell clones. No modification of cytoskeletal integrity was observed (Fig. 3.C). Structure of other filaments like tubulin and vimentin was also not modified (data not shown). Biochemical post-translational modi- fications of cytoskeleton components were also analyzed. We first analyzed the acetylated form of a-tubulin. This modified tubulin is present in various microtubule structures where it acts as a stabilizer essential in many pathways like cell growth, cell migration and morphogenesis [31]. As revealed by immunoblot- ing, a-tubulin was hyperacetylated in Hsp27 depleted HeLa cells (Fig. 3.D). This observation prompted us to analyze histone deacetylase 6 (HDAC6), the mammalian enzyme responsible of a- tubulin deacetylation [31]. As seen in Fig. 3.D, in Hsp27 depleted HeLa cells, the level of HDAC6 polypeptide was dramatically decreased in a negative correlation with a-tubulin hyperacetyla- tion (Fig. 3.D). Similar increase in a-tubulin hyperacetylation was observed when HDAC-6 level was artificially down regulated with a specific shRNA-HDAC6 (Fig. 3.D). This last phenomenon occurred without altering the level of Hsp27. The same experiments were performed in MCF-7 cells that contain a high level of HDAC6. Only a slight decrease of this protein was observed in MSh27-1.3 cells (Fig. 3.D); a phenomenon that corresponded to the small decrease in Hsp27 level and to no significant increase in a-tubulin acetylation and nucleus aberration phenotype. To better characterize the mechanism that generates, in HeLa cells, HDAC6 protein depletion in absence of Hsp27, we performed a quantification of hdac6 gene product by quantitative PCR (qPCR). As shown in Fig. 3.E, the level of HDAC6 mRNA was not significantly altered in HSh27-2.2 cells compared to HMs27 cells. In order to determine if the level of HDAC6 protein was post-translationaly regulated, HeLa cells where treated with PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e29719