STAT2, Caspase3 and HDAC6 as Hsp27 Client Proteins Figure 4. Knock out of Hsp27 induces procaspase-3 proteasomal degradation without modulating the expression of its gene. A, Total extracts of HeLa cell clones were analyzed in immunoblots incubated and revealed with caspase-3 antibody. Quantification of blots intensity was reported in a table. B, Each cell line was treated for 4 h with 0.25 mmol/L of staurosporine. Samples were analyzed in blot using anti caspase-3 antibody. C, Quantification of procaspase-3 gene relative expression by qPCR experiments. D, Effect of proteolytic inhibitors. Samples were collected after being treated with MG132 or ALLN, as described in Fig. 3. Levels of procaspase-3 were revealed by western blot analysis. doi:10.1371/journal.pone.0029719.g004 depleted cells, a DNA vector containing an ISRE element fused to luciferase reporter gene [36] was transfected in control and Hsp27 depleted HeLa cells. Cells were treated with interferon-a (50 U/ml) to activate the STAT2 interferon response. As seen in Fig. 5.F, luciferase expression was down regulated in HSh2.2 cells as compared to control cells, suggesting a decrease in STAT2 transcriptional activity. Moreover, we used two phosphorylation inhibitors, SB203580 and SB202190 (p38MAPkinase and MK2 inhibitors, respectively) to analyze the role of Hsp27 phosphorylation. Treatment with these inhibitors decreased STAT2 transcriptional activity (Fig. 5.F), hence suggesting a crucial role for Hsp27 and its phosphorylation in the regulation of STAT2 response to interferon-a. STAT2, HDAC6 and procaspase3 directly interact with Hsp27 In accordance with the ‘‘holdases’’ and ‘‘molecular sponges’’ theories [4,37], we hypothesized that the chaperone activity of Hsp27 can stabilize polypeptides and modulates their half-life. The chaperone activity of Hsp27 is highly correlated with its structural organization. Indeed, this protein can form dynamic oligomeric structures up to 800 kDa [4,38]. Hsp27 oligomerization profiles were then analyzed by the use of size exclusion columns (Fig. 6.A). In non-stressed cells, Hsp27 protein was currently present in two oligomeric populations with distinct native sizes, the small oligomers (#200 kDa) and the large oligomeric structures over 200 kDa. Immunoblot analysis of the different eluted fractions revealed that procaspase-3 was only present in the fractions corresponding to Hsp27 population of small native sizes whereas STAT2 and HDAC6 proteins were only identified in higher, but distinct, molecular weights fractions (Fig. 6.A). Physical interac- tions between Hsp27 and procaspase3, HDAC6 and STAT2 were tested by Co-IP experiments on column fractions. Goat polyclonal antibody raised against Hsp27 was added to pooled column PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e29719