STAT2, Caspase3 and HDAC6 as Hsp27 Client Proteins Figure 3. HDAC6 is degraded in Hsp27 deficient cell lines. A, photographs of nuclei of Hsp27 depleted HeLa cells stained by Hoechst. B, phase contrast pictures of a depleted HeLa clone showing a giant cell containing more than twenty nuclei. Bar = 10 mm. C, Fluorescence photomicrographs analysis of Hsp27 and F-actin. HeLa cells were plated on glass cover slips and allowed to enter exponential growing phase for one day. Thereafter, cells were fixed and exposed to Hsp27 mouse monoclonal antibody followed by a goat anti-mouse FITC-conjugated antibody. Cells were also treated with TRITC-labeled phalloidin to visualize F-actin and with Hoechst to detect nuclei. Cells were then examined under a fluorescent microscope and photomicrographs including overlay analysis were recorded. Note the drastic modification of cells morphology induced by Hsp27 withdrawal. Bar = 10 mm. D, Transient transfection with ShRNA-HDAC6 and MsRNA-HDAC6 encoding vectors as a control of tubulin induced hyperacetylation. Cell samples were collected and analyzed by immunobloting probed with the indicated antibodies. Quantification of western blot was reported on a table. E, quantification of hdac6 gene relative expression by qPCR analysis. F, proteasome activity, blocked by a low dose of MG132 (0.5 mmol/l- 20 hours), inversely correlates with poly-ubiquitin accumulation, as revealed by immunoblot analysis. Cells were submitted to 20 h treatments with either 10 mmol/L ALLN or 0.5 mmol/L MG132, calpain and proteasome inhibitors, respectively. Levels of HDAC6 were revealed by immunobloting. doi:10.1371/journal.pone.0029719.g003 Hsp27 constitutive depletion induces STAT2 degradation and modulates STAT3 phosphorylation Hsp27 has been reported to interact with STAT3, a crucial transcription factor implicated in the maintenance and anti- apoptotic status of cancerous cells by activating the expression of genes like Bcl-XL and survivin [33,34]. We then tested whether Hsp27 could interact with other member of the STAT family protein. As revealed by western blot analysis, the level of STAT2 was dramatically decreased in Hsp27 depleted HeLa clones (Figure 5.A). In contrast, other proteins of the STAT transcription factor family were not or only weakly (STAT4) modulated (Fig. 5.A). STAT2 transcription factor is implicated in processes such as viral or interferon responses and was not previously described to interact with Hsp27 [35]. Quantification of immunoblots performed with protein extracts from MCF-7 cell lines confirmed the results observed in HeLa cells, since STAT2 endogenous level decreased of 18.2% and 9,8% in the two MCF-7 partially depleted clones, respectively (Figure 5.B). A preceding study has reported the depletion of STAT3 in prostate cells when Hsp27 was transitory decreased by antisense strategy [21]. In our conditions of constitutive depletion of Hsp27, endogenous level of STAT3 remained constant. STAT3 mRNA level was also not altered in Hsp27 depleted clones (Data S1.A). In contrast, STAT3 phosphorylation, induced by interleukin-6 (IL-6) or heat-shock at 43uC, was decreased in correlation to Hsp27 level. A confirmation of Rocchi et al. results was nevertheless obtained in transient transfection experiments of Sh27, suggesting a possible adaptation mechanism to the lack of Hsp27 in our constitutive clones (Data S1.B). As proven by qPCR experiments, STAT2 messenger level was slightly increased in the characterized clones although the level of STAT2 polypeptide was reduced (Fig. 5.D). Furthermore, degradation of STAT2 was blocked when proteolytic degradation was inhibited by MG132 and ALLN treatments, implicating the crucial role of Hsp27 in the stability of this protein (Fig. 5.E). STAT2 is a major transcription factor implicated in the interferon response [35]. To analyze its transcriptional activation in Hsp27 PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e29719